Electrolyte and non-electrolyte distribution in the ehrlich ascites tumor cells during the cell cycle

In a previous study, evidence was presented for changes in the state of water and osmotically active solutes during the cell cycle. Total water was constant at 82% (w/w), while the fraction of water that was osmotically active decreased from a maximum during S to a minimum at mitosis. Total Na⁺, K⁺, and C1^? in milliequivalents per liter of cell water remained constant. Therefore, electrolytes are sequestered in the osmotically inactive water. Evidence is now presented that Na⁺ exists primarily as one compartment, with a second, slower compartment appearing during S and disappearing during G2. Na⁺ is completely exchangeable during the entire cell cycle. The distribution of other penetrating solutes was also investigated. When placed in hyperosmotic ethylene glycol solutions, cells first shrink, then swell to their original volumes. ¹⁴C-ethylene glycol distributes in 89% of cell water throughout the cell cycle. However, ¹⁴C-urea distributes in anywhere from 86–100% of the cell water, depending on the stage in the cell cycle. Both solutes are at chemical equilibrium in water in which they are distributed, but they differ in their effects on cell volume. The final volume at which cells equilibrate in urea varies with the concentration of urea in the environment and with time into the cell cycle. Results suggest a loss of osmotically active particles or decreased osmotic activity of urea.     

MAINTENANCE OF IONS,PROTEINS AND WATER IN LENS FIBER CELLS BEFORE AND AFTER TREATMENT WITH NON-IONIC DETERGENTS

If the plasma membrane and its associated transport proteins are solely responsible for maintenance of the asymmetric solute distribution then disruption of the plasma membrane would quickly lead to the symmetric distribution of all unattached inorganic ions between the cell and the extracellular environment. To test this hypothesis fresh pig lenses were incubated in Hanks ’ balanced salt solution in either absence or presence of non-ionic detergents (0.2 % Triton X-100 or 0.2 % Brij 58). Both detergents caused permeabilization of every lens fiber cell as shown by electron microscopy. The flux kinetics of K⁺, Mg^{2 +}, Na⁺, Ca^{2 +}, water and protein out of and into the permeabilized lens fiber cells was measured. Triton X-100 caused a faster flux rate of all solutes than did Brij 58. The Triton X-100 induced flux of solutes and water was associated with a decrease in lens ATP. Incubation of untreated lenses in solutions of different osmotic pressures at 0 °C demonstrated that the major fraction of lens water was osmotically unresponsive. Thus the asymmetric distribution of solutes in lens fiber cells is dependent on an intact plasma membrane and on a co-operative ATP-dependent association between K⁺, Mg^{2 +}, water and cytomatrix proteins.     

Isolated epithelial cells of the toad bladder. Their preparation, oxygen consumption, and electrolyte content

Epithelial cells of the toad bladder were disaggregated with EDTA, trypsin, hyaluronidase, or collagenase and were then scraped free of the underlying connective tissue. In most experiments EDTA was complexed with a divalent cation before the tissue was scraped. Q _OO2, sucrose and inulin spaces, and electrolytes of the isolated cells were measured. Cells disaggregated by collagenase or hyaluronidase consumed O₂ at a rate of 4 µl hr^-1 dry wt^-1. Q _OO2 was increased 50% by ADH (100 U/liter) or by cyclic 3'',5''-AMP (10 mM/liter). Na⁺-free Ringer''s depressed the Q _OO2 by 40%. The Q _OO2 of cells prepared by trypsin treatment or by two EDTA methods was depressed by Na⁺-free Ringer''s but was stimulated relatively little by ADH. Two other EDTA protocols produced cells that did not respond to Na⁺ lack or ADH. The intracellular Na⁺ and K⁺ concentrations of collagenase-disaggregated cells were 32 and 117 mEq/kg cell H₂O, respectively. Cation concentrations of hyaluronidase cells were similar, but cells that did not respond to ADH had higher intracellular Na⁺ concentrations. Cells unresponsive to ADH and Na⁺ lack had high sucrose spaces and low transcellular membrane gradients of Na⁺, K⁺, and Cl^-. The results suggest that trypsin and EDTA disaggregation damage the active Na⁺ transport system of the isolated cell. Certain EDTA techniques may also produce a general increase in permeability. Collagenase and hyaluronidase cells appear to function normally.     

Sodium, Potassium, and Chloride Fluxes in Intercostal Muscle from Normal Goats and Goats with Hereditary Myotonia

In isolated bundles of external intercostal muscle from normal goats and goats with hereditary myotonia the following were determined: concentrations and unidirectional fluxes of Na⁺, K⁺, and Cl^-, extracellular volume, water content, fiber geometry, and core-conductor constants. No significant difference between the two groups of preparations was found with respect to distribution of fiber size, intracellular concentrations of Na⁺ or Cl^-, fiber water, resting membrane potential, or overshoot of action potential. The intracellular Cl^- concentration in both groups of preparations was 4 to 7 times that expected if Cl^- were distributed passively between intracellular and extracellular water. The membrane permeability to K (P_K) calculated from efflux data was (a) at 38°C, 0.365 x 10^-6 cm sec^-1 for normal and 0.492 x 10^-6 for myotonic muscle, and (b) at 25°C, 0.219 x 10^-6 for normal and 0.199 x 10^-6 for myotonic muscle. From Cl^- washout curves of normal muscle usually only three exponential functions could be extracted, but in every experiment with myotonic muscle there was an additional, intermediate component. From these data PP_cl could be calculated; it was 0.413 x 10^-6 cm sec^-1 for myotonic fibers and was 0.815 x 10^-6 cm sec^-1 for normal fibers. The resting membrane resistance of myotonic fibers was 4 to 6 times greater than that of normal fibers.     

Response of performance and bacterial community to oligotrophic stress in biofilm systems for raw water pretreatment

Understanding the dynamics of performance and bacterial community of biofilm under oligotrophic stress is necessary for the process optimization and risk management in biofilm systems for raw water pretreatment. In this study, biofilm obtained from a pilot-scale biofilm reactor was inoculated into a pilot-scale experimental tank for the treatment of oligotrophic raw water. Results showed that the removal of NH₄ ⁺–N was impaired in biofilm systems when influent NH₄ ⁺–N was less than 0.35 mg L^?1 or NH₄ ⁺–N loading rate of less than 7.51 mg L^?1 day^?1. The dominant bacteria detected in biofilm of different carrier were obvious distinct from phylum to genus level under oligotrophic stress. The dominant bacteria in elastic stereo media carrier changed from Proteobacteria (51.1%) to Firmicutes (32.7%), while Proteobacteria was always dominant in suspended ball carrier after long-term operation under oligotrophic conditions. Oligotrophic stress largely decreased the functional bacteria for the removal of nitrogen and organics including many genera in Proteobacteria and Nitrospirae, but increased several genera with spore forming organisms or potential bacterial pathogens in ESM carrier mainly including Bacillus, Mycobacterium, Pseudomonas, etc.     

Solvent effects on intramolecular hydrophobic ligandligand interactions in binary and ternary complexes

The stability constants of mixed ligand complexes of the type M(Phen)(ACA)⁺, where M = Cu²⁺ or Zn²⁺, Phen = 1,10-phenanthroline and ACA^? = propionate, valerate and 2-cyclohexylacetate, were determined by potentiometric pH titration in 50% (v/v) dioxanewater and were compared with the stabilities of the corresponding ternary complexes formed with formate and acetate. The ternary complexes containing the alkanecar?ylates (ACA^?) are significantly more stable, due to intramolecular hydrophobic interactions between the alkyl residue of the ACAt¯ligands and the 1,10-phenanthroline molecule. For Zn(Phen)(valerate)⁺ this intramolecular ligand-ligand interaction was confirmed by¹H NMR shift measurements. The formation degree of the intramolecular adducts in the ternary Cu²⁺ and Zn²⁺ complexes was calculated and the position of the intramolecular equilibrium between the opened and closed isomer was determined: the closed isomer occurs between about 10 to 35 percent. Comparisons with related data show that the extent of this interaction is about the same in water and in 50% aqueous dioxane; this contrasts with the experience made with simple unbridged adducts, which are destabilized by the addition of dioxane (or other organic solvents). Furthermore, evaluation of the available stability data for the Cu²⁺/leucinate (Leu^?) system shows that addition of some dioxane to an aqueous solution (in which of the closed isomer exists to about 20%) favors the intramolecular interaction between the two isopropyl residues in Cu(Leu)₂ considerably: in 40 to 50% aqueous dioxane the formation degree of the closed isomer reaches about 80%. Higher concentrations of the organic solvent destabilize the hydrophobic interaction. The overall stability of Cu(Leu)⁺ and Cu(Leu)₂, as well of Cu(alaninate)⁺ and Cu(alaninate)₂, is governed by the polarity of the solvent while the extent of the intramolecular ligand-ligand interaction is influenced by the hydrophobic properties of the solvent molecules. Based on the stability data it is shown that intramolecular ligandligand interactions are quite a common feature for many binary and ternary amino acid complexes: e.g., M(norvalinate)₂, M(phenyl-alaninate)₂, M(tyrosinate)₂ [M = Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺] or Cu(tyrptophanate)₂ and M(phenylalaninate)(norvalinate) or M(phenylalaninate)(tyrosinate) [M = Co²⁺, Ni²⁺, Cu²⁺]. In addition, evidence is presented that direct M²⁺-aromatic interactions are of no significance in these amino acid complexes in solution. The relevance of the present results with regard to biological systems is indicated.     

On the Mechanism of Sodium Extrusion across the Irrigated Gill of Sea Water-Adapted Rainbow Trout (Salmo gairdneri)

Sodium efflux (J_out^Na) across the irrigated trout gill was rapid in sea water (SW), but only about 25 % as large in fresh water (FW). The difference correlated with a change in the potential difference across the gill (TEP). The latter was about +10 mV (blood positive) in SW, but –40 mV in FW. Both flux and electrical data indicated that gills in this fish are permeable to a variety of cations including Na⁺, K⁺, Mg²⁺, choline, and Tris. They are less permeable to anions; P_Na:P_K:P_Cl was estimated to be 1:10:0.3, and P_Cl > P_gluconate. The TEP was shown to be a diffusion potential determined by these permeabilities and the extant ionic gradients in SW, FW as well as in other media. J_out^Na appeared to be diffusive in all of the experiments undertaken. Exchange diffusion need not be posited, and the question of whether there is an active component remains open.     

New chiral macrocyclic lanthanide complexes derived from (1R,2R)-1,2-diaminocyclohexane and 2,6-diformylpyridine

The new enantiopure complexes [LnL](NO₃)₃ · nH₂O (Ln = Dy⁺³, Ho⁺³, Er⁺³, Lu⁺³) and [LnL]Cl₃ · nH₂O (Ln = Nd⁺³, Sm⁺³, Gd⁺³, Tb⁺³, Dy⁺³, Ho⁺³, Er⁺³, Tm⁺³, Lu⁺³) of the chiral macrocycle L derived from (1R,2R)-1,2-diaminocyclohexane and 2,6-diformylpyridine have been synthesised. The preference of macrocycle L for the heavier lanthanide(III) ions has been established on the basis of competition reaction. The complexes have been characterised by NMR spectroscopy and mass spectrometry. ¹H NMR signals of deuterated water solutions of the Ce⁺³, Nd⁺³ and Eu⁺³ complexes have been assigned on the basis of the COSY and HMQC spectra, and for the remaining lanthanide complexes the signals were assigned on the basis of linewidths analysis. The paramagnetic shifts of the series of lanthanide complexes [LnL](NO₃)₃ · nH₂O and [LnL]Cl₃ · nH₂O have been analysed using both crystal-field dependent and independent methods in order to separate contact and dipolar contributions and establish isostructurality along the series of lanthanide complexes in solution. The data obtained for nitrate derivatives in organic solvent indicate rather irregular deviations from the plots based on those methods, while the plots obtained for water solutions show the characteristic brake in the middle of the lanthanide series, that is interpreted as a result of change of the number of axially coordinated water molecules. The apparent inconsistencies of results obtained on the basis of crystal-field independent method are discussed.     

The Anisotropic Elastic Properties of the Sarcolemma of the Frog Semitendinosus Muscle Fiber

Tension and curvature of the sarcolemmal tube of the frog muscle fiber were measured at different extensions and were used to calculate the anisotropic elastic properties of the sarcolemma. A model was derived to obtain the four parameters of the elasticity matrix of the sarcolemma. Sarcolemmal thickness was taken as 0.1 μm. Over the range of reversible sarcolemmal tube extension, the longitudinal elastic modulus E_L = 6.3 × 10⁷ dyn/cm², the circumferential modulus E_c = 0.88 × 10⁷ dyn/cm², the longitudinal Poisson's ratio σ_L = 1.2, and the circumferential Poisson's ratio σ_c = 0.18. At tubular rest length E_L = 1.2 × 10⁷ dyn/cm². The sarcolemma is less extensible in the longitudinal direction along the fiber axis than in the circumferential direction. It can be extended reversibly to 48% of its rest length, equivalent to extending the intact fiber from a sarcomere length of 3 μm to about 4.5 μm. The sarcolemma does not contribute to intact fiber tension at fiber sarcomere lengths <3 μm, and between 3 and 4 μm its contribution is about 20%. It also exerts a pressure on the myoplasm, which can be calculated by means of the model. The longitudinal elastic modulus of the whole fiber is 1 × 10⁵ dyn/cm² at a sarcomere length of 2.33 μm.     

Cell ultrastructure and fatty acid composition of lipids in vegetative organs of <Emphasis Type="Italic">Chenopodium album</Emphasis> L. under salt stress conditions

White goosefoot plants (Chenopodium album L. of the family Chenopodiaceae) grown at various NaCl concentrations (3–350 mM) in the nutrient solution were used to study the cell ultrastructure as well as the qualitative and quantitative composition of fatty acids in the lipids of vegetative organs. In addition, the biomass of Ch. album vegetative organs, the water content, and the concentrations of K⁺, Na⁺, and Cl^– were determined. The growth rates of plants raised at NaCl concentrations up to 200–250 mM were the same as for the control plants grown at 3 mM NaCl; the growth parameters remained rather high even at NaCl concentrations of 300–350 mM. The water content in Ch. album organs remained high at all NaCl concentrations tested. Analysis of the ionic status of Ch. album revealed a comparatively high K⁺ content in plant organs. At low NaCl concentrations in the nutrient solution, K⁺ ions were the dominant contributors to the osmolarity (the total concentration of osmotically active substances) and, consequently, to the lowered cell water potential in leaves and roots. As the concentration of NaCl was increased, the plant organs accumulated larger amounts of Na⁺ and Cl^–, and the contribution of these ion species to osmolarity became increasingly noticeable. At 300–350 mM NaCl the contribution of Na⁺ and Cl^– to osmolarity was comparable to that of K⁺. An electron microscopy study of Ch. album cells revealed that, apart from the usual response to salinity manifested in typical ultrastructural changes of chloroplasts, mitochondria, and the cytosol, the salinity response comprised the enhanced formation of endocytic structures and exosomes and stimulation of autophagy. It is supposed that activation of these processes is related to the removal from the cytoplasm of toxic substances and the cell structures impaired by salt stress conditions. The qualitative and quantitative composition of fatty acids in the lipids of Ch. album organs was hardly affected by NaCl level. These findings are consistent with the high salt tolerance of Ch. album, manifested specifically in retention of growth functions under wide-range variations of NaCl concentration in the nutrient solution and in maintenance of K⁺, Na⁺, and Cl^– content in organs at a constant level characteristic of untreated plants.     

Osmotic stress and viscous retardation of the Na,K-ATPase ion pump

The transport function of the Na pump (Na,K-ATPase) in cellular ion homeostasis involves both nucleotide binding reactions in the cytoplasm and alternating aqueous exposure of inward- and outward-facing ion binding sites. An osmotically active, nonpenetrating polymer (poly(ethyleneglycol); PEG) and a modifier of the aqueous viscosity (glycerol) were used to probe the overall and partial enzymatic reactions of membranous Na,K-ATPase from shark salt glands. Both inhibit the steady-state Na,K-ATPase as well as Na-ATPase activity, whereas the K⁺-dependent phosphatase activity is little affected by up to 50% of either. Both Na,K-ATPase and Na-ATPase activities are inversely proportional to the viscosity of glycerol solutions in which the membranes are suspended, in accordance with Kramers’ theory for strong coupling of fluctuations at the active site to solvent mobility in the aqueous environment. PEG decreases the affinity for Tl⁺ (a congener for K⁺), whereas glycerol increases that for the nucleotides ATP and ADP in the presence of NaCl but has little effect on the affinity for Tl⁺. From the dependence on osmotic stress induced by PEG, the aqueous activation volume for the Na,K-ATPase reaction is estimated to be ∼5-6 nm³ (i.e., ∼180 water molecules), approximately half this for Na-ATPase, and essentially zero for p-nitrophenol phosphatase. The change in aqueous hydrated volume associated with the binding of Tl⁺ is in the region of 9 nm³. Analysis of 15 crystal structures of the homologous Ca-ATPase reveals an increase in PEG-inaccessible water space of ∼22 nm³ between the E₁-nucleotide bound forms and the E₂-thapsigargin forms, showing that the experimental activation volumes for Na,K-ATPase are of a magnitude comparable to the overall change in hydration between the major E₁ and E₂ conformations of the Ca-ATPase.     

Essential sulfhydryl group of malic enzyme fromEscherichia coli

The activity of malic enzyme fromEscherichia coli was unaffected by the monovalent cations Na⁺ or Li⁺ at 10 mM. At 100 mM, Li⁺ or Na⁺ inhibited the enzyme activity by 88% and 83%, respectively. However, the enzyme activity was stimulated by 40–80-fold with 10 mM K⁺, Rb⁺, Cs⁺, or NH ₄ ⁺ . Less stimulation was observed with 100 mM of these stimulating cations. The stimulatory effect was lost after the enzyme was dialyzed against Tris-Cl buffer, but was regained after incubating the dialyzed enzyme with dithiothreitol. The regenerated enzyme was inactivated by 5,5′-dithiobis(2-nitrobenzoic acid). The resulting inactive thionitrobenzoyl enzyme could be regenerated to the active thiol-enzyme by eithiothreitol or converted to the inactive thiocyanoylated enzyme by KCN. The thiocyanoylated enzyme was insensitive to K⁺ stimulation, which suggested the essentiality of the sulfhydryl groups of theE. coli malic enzyme.     

Prostaglandin E2 enhances the sodium conductance of exocrine glands in isolated frog skin (Rana esculenta)

Summary Prostaglandins are known to stimulate the active transepithelial Na⁺ uptake and the active secretion of Cl^– from the glands of isolated frog skin. In the present work the effect of prostaglandin E₂ (PGE₂) on the glandular Na⁺ conductance was examined. In order to avoid interference from the Na⁺ uptake and the glandular Cl^– secretion the experiments were carried out on skins where the Cl^– secretion was inhibited (the skins were bathed in Cl^– Ringer's solution in the presence of furosemide, or in NO ₃ ^– Ringer's solution), and the active Na⁺ uptake was blocked by the addition of amiloride. Transepithelial current, water flow and ion fluxes were measured. A negative current was passed across the skins (the skins were clamped at –100 mV, basolateral solution was taken as reference). When PGE₂, was added to the skins under these experimental conditions, the current became more negative; this was mainly due to an increase in the Na⁺ efflux. Together with the increase in Na⁺ efflux a significant increase of the water secretion was observed. The water secretion was coupled to the efflux of Na⁺, and when one Na⁺ was pulled from the basolateral to the apical solution via this pathway 230 molecules of water follwed. From the data presented it is suggested that this pathway for Na⁺ is confined to the exocrine glands.     

Ammonium nutrition increases water absorption in rice seedlings (Oryza sativa L.) under water stress

Water stress is a primary limitation on plant growth. In previous studies, it has been found that ammonium enhances the tolerance of rice plants to water stress, but how water is related to nitrogen form and water stress remains unknown. To study the effects of nitrogen form (NH ₄ ⁺ , NO ₃ ^? , and a mixture of NH ₄ ⁺ and NO ₃ ^? ) on the growth and water absorption of rice (Oryza sativa L.) seedlings, a hydroponic experiment with water stress, simulated by the addition of polyethylene glycol (PEG, 10% w/v, MW 6000), was conducted in a greenhouse. The results showed that, compared with non-water stress, under water stress, the fresh weight of rice seedlings increased by 14% with NH ₄ ⁺ nutrition, whereas it had decreased by about 20% with either NO ₃ ^? or mixed nitrogen nutrition. No significant difference was found in the transpiration rate of excised shoots or in xylem exudation of excised roots in NH ₄ ⁺ supply between the two water situations, whereas xylem flow decreased by 57% and 24% under water stress in NO ₃ ^? and mixed nutrition, and root hydraulic conductivity decreased by 29% and 54% in plants in NH ₄ ⁺ and NO ₃ ^? nutrition conditions, respectively. Although water absorption ability decreased in both NH ₄ ⁺ and NO ₃ ^? nutrition, aquaporin activity was higher in NH ₄ ⁺ than in NO ₃ ^? nutrition, regardless of water stress. We conclude that NH ₄ ⁺ nutrition can improve water handling in rice seedlings and subsequently enhance their resistance to drought.     

Density functional theoretical study on the preferential selectivity of macrocyclic dicyclohexano-18-crown-6 for Sr+2 ion over Th+4 ion during extraction from an aqueous phase to organic phases with different dielectric constants

The preferential selectivity of dicyclohexano-18-crown-6 (DCH18C6) for bivalent Sr⁺² ion over tetravalent Th⁺⁴ ion was investigated using generalized gradient approximated (GGA) BP86 and the hybrid B3LYP density functional, employing split valence plus polarization (SV(P)) and triple-zeta valence plus polarization (TZVP) basis sets in conjunction with the COSMO (conductor-like screening model) solvation approach. The calculated theoretical selectivity of DCH18C6 for Sr⁺² ion over Th⁺⁴ ion was found to be in accord with the selectivity for Sr⁺² ion over Th⁺⁴ ion observed when performing liquid–liquid extraction experiments in different organic solvents. While 1:1(M:L) stoichiometric complexation reactions can be used to predict the preferential selectivity of Sr²⁺ ion over Th⁴⁺ ion, the results obtained are not consistent with the experimental results observed upon increasing the dielectric constant of the solvent. The calculated theoretical gas-phase data for the free energy of complexation, ?G, fail to explain the selectivity for Sr⁺² ion over Th⁺⁴ ion. However, when 1:2 (M:L) stoichiometric complexation reactions (reported in previous X-ray crystallography studies) are considered, correct and consistent results for the selectivity for Sr⁺² ion over a wide range of dielectric constants are predicted. The distribution constant for Sr²⁺ and Th⁴⁺ ions was found to gradually increase with increasing dielectric constant of the organic solvent, and was found to be highest in nitrobenzene. The selectivity data calculated from ??G _ext are in excellent agreement with the results obtained from solvent extraction experiments.

Decrease in the electrogenic Contribution of Na,K-ATPase and the resting membrane potential as a possible mechanism of Ca<Superscript>2+</Superscript> accumulation in rat soleus muscle in a short-term gravity unloading

The resting membrane potential and electrogenic contribution of α1- and α2-isoforms of Na⁺/K⁺-ATPase in the rat soleus muscle at early stages of gravity unloading were analyzed. The role of L-type calcium channels in accumulation of calcium ions in the myoplasm under these conditions was estimated. After 3-day antiorthostatic suspension, the resting membrane potential of the muscle fibers decreased from ?71.0 ± 0.5 to ?66.8 ± 0.7 mV, the muscle excitability reduced, and a trend of muscle fatigue acceleration appeared. The electrogenic contribution of ouabain-sensitive α2-isoform of Na⁺/K⁺-ATPase, determined as the depolarization caused by 1μM ouabain, decreased after suspension from 6.2 ± 0.6 to 0.5 ± 0.8 mV. The contribution of ouabain-resistant α1-isoform of Na⁺/K⁺-ATPase, determined as an additional depolarization after addition of 500 μM ouabain, decreased from 4.6 ± 0.6 to 2.6 ± 0.6 mV. The intensity of Fluo-4AM fluorescence in individual muscle fibers increased after suspension more than fourfold, which suggests an elevated calcium concentration in the myoplasm. A local delivery of nifedipine, a blocker of the L-type calcium channels, to the muscle removed this effect. The existence of a selective mechanism suppressing the electrogenic contribution of Na⁺/K⁺-ATPase α2-isoform, which is the main cause of the muscle fiber membrane depolarization after 3-day suspension, is postulated. The depolarization can activate part of potential-sensitive L-type Ca²⁺ channels, causing the accumulation of calcium ions in the muscle fiber myoplasm.     

Estimating the contribution of arbuscular mycorrhizal fungi to drought tolerance of potted olive trees (<Emphasis Type="Italic">Olea europaea</Emphasis>)

The aim of the present study is to investigate the contribution of mycorrhization to the resilience of olive trees to drought. One-year-old olive plants were inoculated (Myc⁺) or not (Myc^?) with arbuscular mycorrhizal fungi (AMF), and subjected to a 40-day-drought period. At regular intervals of the watering-off period and after rehydration period, water relations and gas exchanges parameters were measured. Similarly, the total soluble sugars, proline, and mineral nutrients concentrations were determined. The results revealed that Myc⁺ plants were less affected by drought than Myc^? plants proving the involvement of the AMF in the alleviation of drought impact on olive tree. In fact, the turgor potential (Ψ_p) in Myc⁺ plants exhibited positive values during the whole treatment period, while Ψ_p in Myc^? plants was negative mainly under severe stress intensity. Moreover, the stomatal function of Myc⁺ plants was less affected by drought compared to Myc^? plants. The maximum of mycorrhizas relative drought alleviation rate (RDAR) was estimated to be 40% for Ψ_pd and RWC, 36% for the osmotic potential (Ψ_S), 86% for Ψ_p, 16% for g_s, and 27% for E. The osmotic adjustment by proline was earlier in Myc⁺ plants than in Myc^? ones. The inoculation with AMF also improved mineral uptake (K, N, Zn, and Fe). After 40 days of drought, Myc⁺ plants survive but not Myc^? ones. In addition, the restoration of the irrigation permitted the Myc⁺ plants to recuperate from severe drought stress. To sum up, inoculation of young olive trees with the AMF improved their resilience to drought.     

Phosphate cycles in energy crop systems with emphasis on the availability of different phosphate fractions in the soil

The growing cells of hydroponic maize roots expand at constant turgor pressure (0.48 MPa) both when grown in low-(0.5 mol m^-3 CaCl₂) or full-nutrient (Hoagland's) solution and also when seedlings are stressed osmotically (0.96 MPa mannitol). Cell osmotic pressure decreases by 0.1–0.2 MPa during expansion. Despite this, total solute influx largely matches the continuously-varying volume expansion-rate of each cell. K⁺ in the non-osmotically stressed roots is a significant exception-its concentration dropping by 50% regardless of the presence or absence of K⁺ in the nutrient medium. This corresponds to the drop in osmotic pressure. Nitrate appears to replace Cl^- in the Hoagland-grown cells.Analogous insensitivity of solute gradients to external solutes is observed in the radial distribution of water and solutes in the cortex 12 mm from the tip. Uniform turgor and osmotic pressures are accompanied by opposite gradients of K⁺ and Cl^-, outwards, and hexoses and amino acids, inwards, for plants grown in either 0.5 mol m^-3 CaCl₂ or Hoagland's solution (with negligible Cl^-). K⁺ and Cl^- levels within both gradients were slightly higher when the ions were available in the medium. The gradients themselves are independent of the direction of solute supply. In CaCl₂ solution all other nutrients must come from the stele, in Hoagland's solution inorganic solutes are available in the medium.24 h after osmotic stress, turgor pressure is recovered at all points in each gradient by osmotic adjustment using organic solutes. Remarkably, K⁺ and Cl^- levels hardly change, despite their ready availability. Hexoses are responsible for some 50% of the adjustment with mannitol for a further 30%. Some 20% of the final osmotic pressure remains to be accounted for. Proline and sucrose are not significantly involved. Under all conditions a standing water potential step of 0.2 MPa between the rhizodermis and its hydroponic medium was found. We suggest that this is due to solute leakage.Abbreviations EDX energy dispersive X-ray microanalysis - water potential - 11-1 cell osmotic pressure - P turgor pressure     

Analytical and experimental studies on the relationship between Na+, K+, and water uptake during volume increases associated with Fundulus oocyte maturation in vitro

Summary Oocytes of marine and estuarine teleosts often undergo pronounced volume increases during the maturation phase of development that precedes ovulation and fertilization. To examine the physiological correlates of these volume increases, prematuration follicles of the saltmarsh teleost, Fundulus heteroclitus, were cultured in vitro with a maturation-inducing steroid (17-hydroxy-20-dihydroprogesterone). Mean follicle volume rose significantly (75%) during a 40-h incubation period. Similar to the situation previously found in vivo, uptake of water by the maturing follicle was responsible for this volume increase in vitro, with the water content increasing from 62% to 78% of the total follicle mass. The follicle contents of two probable osmotic effectors-Na⁺ and K⁺-also rose, the increase in K⁺ being twice that of Na⁺. The influx of K⁺ even exceeded water uptake, resulting in a net increase in the concentration of this cation. It thus appears that the influx of these cations, in particular K⁺, is a major cause of the uptake of osmotically obligated water and subsequent volume increase experienced by maturing F. heteroclitus follicles. In a search for operant mechanisms, it was found that follicle hydration, but not maturation, was strictly dependent on external K⁺ in a concentration-dependent manner. The mechanism by which K⁺ accumulates in the follicle was insensitive to ouabain, so that a typical Na⁺, K⁺-ATPase mechanism does not appear to be involved. The ability of external K⁺ to promote follicle hydration was gradually lost during the maturation process as the oocyte dissociated from the surrounding granulosa cells in preparation for ovulation. Removal of all associated somatic cells prior to maturation prevented subsequent steroid-initiated hydration but not maturation. The results suggest that K⁺ may be translocated from surrounding granulosa cells to the oocyte via gap junctions during maturation.Abbreviations GVBD germinal vesicle breakdown     

Identification and Characterization of the Na+/H+ Antiporter Nhas3 from the Thylakoid Membrane of Synechocystis sp. PCC 6803

Na⁺/H⁺ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na⁺/H⁺ antiporters, nhaS1–5 and sll0556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na⁺/H⁺ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na⁺ concentrations. NhaS3 had no K⁺/H⁺ exchange activity itself but enhanced K⁺ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO₂ to high CO₂ conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO₂ consumption caused by the ceasing of O₂-evolving photosynthesis. This is the first report of a Na⁺/H⁺ antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H⁺, Na⁺, and K⁺ in supporting the conversion of photosynthetic products and in the supply of energy in the dark.Na⁺/H⁺ antiporters are integral membrane proteins that transport Na⁺ and H⁺ in opposite directions across the membrane and that occur in virtually all cell types. These transporters play an important role in the regulation of cytosolic pH and Na⁺ concentrations and influence proton or sodium motive force across the membrane (1, 2). In Escherichia coli, three Na⁺/H⁺ antiporters (NhaA, NhaB, and ChaA) have been described in detail. Of these, NhaA is the functionally best characterized transporter. The crystal structure of NhaA has been resolved (3). In addition, mutants of nhaA, nhaB, and chaA as well as the triple mutant have been generated (4). The triple mutant was shown to be hypersensitive to extracellular Na⁺. The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains six genes encoding Na⁺/H⁺ antiporters (NhaS1–5 and sll0556). NhaS1 (slr1727) has also been designated SynNhaP (5, 6). Null mutants of nhaS1, nhaS2, nhaS4, and nhaS5 have been generated; however, a null mutant of nhaS3 could not be obtained, indicating that it is an essential gene (6–8). By heterologous expression in E. coli, Na⁺/H⁺ exchange activities could be shown for NhaS1–5 (5, 6). Inactivation of nhaS1 and nhaS2 results in retardation of growth of Synechocystis (5, 6). It has been reported that in these mutants the concentration of Na⁺ in cytosol and intrathylakoid space (lumen) increases and impairs the photosynthetic and/or respiratory activity of the cell (9, 10). Therefore the Na⁺ extrusion by Synechocystis Na⁺/H⁺ antiporters similar to E. coli NhaA, NhaB, and ChaA is essential for the adaptation to salinity stress.In contrast to the case in E. coli, Na⁺ is an essential element for the growth of some cyanobacteria (11, 12). Interestingly, the Na⁺/H⁺ antiporter homolog NhaS4 was identified as an uptake system for Na⁺ from the medium during a screen for mutations in Synechocystis that result in lack of growth at low Na⁺ concentrations (7). The requirement of a Na⁺ uptake antiporter for cell growth is consistent with the physiology of Synechocystis. Specifically, photoautotrophic bacteria like cyanobacteria share some components (plastoquinone, cytochrome b₆f, and c₆) of the thylakoid membrane for electron transport for both photophosphorylation and respiratory oxidative phosphorylation. Na⁺/H⁺ antiporters therefore may coordinate both H⁺ and Na⁺ gradients across the plasma and thylakoid membranes to adapt to daily environmental changes (11). It remains to be determined whether the six Na⁺/H⁺ antiporters are localized to the plasma membrane or to the thylakoid membrane in Synechocystis. Information on the membrane localization will also provide information on the physiological role in Synechocystis. In this study, we explored the membrane localization of NhaS3, the role of specific amino acid residues for its function, and the effect of CO₂ concentration and circadian rhythms on the expression pattern of nhaS3 to gain insight into the physiological role of NhaS3 in Synechocystis.