Cation conductance and efflux induced by polyene antibiotics in the membrane of skeletal muscle fiber.

Cation conductance and efflux induced by polyene antibiotics amphotericin B (AMB), amphotericin B methyl ester (AME), nystatin, mycoheptin, and levorin on frog isolated skeletal muscle fibers and whole sartorius muscles were investigated. Conductance was measured under current-clamp conditions using a double sucrose-gap technique. Cation efflux was studied using flame emission photometry. Some new data were obtained concerning the effects of levorin and mycoheptin on biological membranes. The power dependence of polyene-induced cation transport on antibiotic concentration in muscle membrane was lower than that in bilayers. The decline in the equilibrium conductance caused by polyene removal (except for levorin) was very fast. There was reverse temperature dependence of AMB- and nystatin-induced conductances. Both induced conductance and efflux values demonstrated a correlation with the order of antifungal activities: levorin > AMB, mycoheptin > AME > nystatin, except for AME, which was more potent on yeastlike cells. These effects were interpreted in terms of possible differences in the kinetics of channel formation in biological and model membranes and in light of the role of nonconducting antibiotic forms in biological membranes.     

Diversity of amyloid beta protein fragment [1-40]-formed channels

1. The lipid bilayer technique was used to characterize the biophysical and pharmacological properties of several ion channels formed by incorporating amyloid beta protein fragment (AP) 1–40 into lipid membranes. Based on the conductance, kinetics, selectivity, and pharmacological properties, the following AP[1–40]-formed ion channels have been identified: (i) The AP[1–40]-formed bursting fast cation channel was characterized by (a) a single channel conductance of 63 pS (250/50 mM KCl cis/trans) at +140 mV, 17 pS (250/50 mM KCl cis/trans) at –160 mV, and the nonlinear current–voltage relationship drawn to a third-order polynomial, (b) selectivity sequence P _K > P _Na > P _Li = 1.0:0.60:0.47, (c) P_o of 0.22 at 0 mV and 0.55 at +120 mV, and (d) Zn²⁺-induced reduction in current amplitude, a typical property of a slow block mechanism. (ii) The AP[1–40]-formed spiky fast cation channel was characterized by (a) a similar kinetics to the bursting fast channel with exception for the absence of the long intraburst closures, (b) single channel conductance of 63 pS (250/50 KCl) at +140 mV 17 pS (250/50 KCl) at –160 mV, the current–voltage relationship nonlinear drawn to a third-order polynomial fit, and (c) selectivity sequence P _Rb > P _K > P _Cs > P _Na > P _Li = 1.3:1.0:0.46:0.40:0.27. (iii) The AP[1–40]-formed medium conductance channel was charcterized by (a) 275 pS (250/50 mM KCl cis/trans) at +140 mV and 19 pS (250/50 mM KCl cis/trans) at –160 mV and (b) inactivation at V_ms more negative than –120 and more positive than +120 mV. (iv) The AP[1–40]-formed inactivating large conductance channel was characterized by (a) fast and slow modes of opening to seven multilevel conductances ranging between 0–589 pS (in 250/50 mM KCl) at +140 mV and 0–704 pS (in 250/50 mM KCl) at –160 mV, (b) The fast mode which had a conductance of <250 pS was voltage dependent. The inactivation was described by a bell-shaped curve with a peak lag time of 7.2 s at +36 mV. The slow mode which had a conductance of >250 pS was also voltage dependent. The inactivation was described by a bell-shaped curve with a peak lag time of 7.0 s at –76 mV, (c) the value of P _K/P _choline for the fast mode was 3.9 and selectivity sequence P _K > P _Cs > P _Na > P _Li = 1.0:0.94:0.87:0.59. The value of P _K/P _choline for the slow mode was 2.7 and selectivity sequence P _K > P _Na > P _Li > P _Cs = 1.0:0.59:0.49:0.21, and (d) asymmetric blockade with 10 mM Zn²⁺-induced reduction in the large conductance state of the slow mode mediated via slow block mechanism. The fast mode of the large conductance channel was not affected by 10 mM Zn²⁺.2. It has been suggested that, although the bursting fast channel, the spiky fast channel and the inactivating medium conductance channel are distinct, it is possible that they are intermediate configurations of yet another configuration underlying the inactivating large conductance channel. It is proposed that this heterogeneity is one of the most common features of these positively-charged cytotoxic amyloid-formed channels reflecting these channels ability to modify multiple cellular functions.3. Furthermore, the formation of -sheet based oligomers could be an important common step in the formation of cytotoxic amyloid channels.     

Diacylglycerols stimulate short-circuit current across frog skin by increasing apical Na+ permeability

Summary The phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) stimulates baseline Na⁺ transport across frog skin epithelium and partially inhibits the natriferic response to vasopressin. The effects are produced largely or solely when TPA is added to the mucosal surface of the tissue. Although TPA activates protein kinase C, it has other effects, as well. Thus, the biochemical basis for the effects and the ionic events involved have been unclear. Furthermore, the physiologic implications have been obscure because of the sidedness of TPA's actions.We now report that two synthetic diacylglycerols (DAG) replicate the stimulatory and inhibitory effects of TPA on frog skin. DAG is the physiologic activator of PKC. In this tissue, it produces half-maximal stimulation at a concentration of 19 M. In contrast to TPA, DAG is about equally effective from either tissue surface.In a series of eight experiments, DAG was found to depolarize the apical membrane. Diacylglycerol also increases the paracellular conductance of frog skins bathed with mucosal Cl^– Ringer's solution. The latter effect can be minimized by replacing NO ₃ ^– for Cl^– in the mucosal solution. Under these conditions, combined intracellular and transepithelial measurements indicated that DAG increased both the apical Na⁺ permeability and intracellular Na⁺ concentration. These results are qualitatively similar to the effects of cyclic 3, 5-AMP on this tissue, suggesting that activation of PKC by DAG causes phosphorylation of the same or nearby gating sites phosphorylated by cAMP.We propose that apical Na⁺ entry is regulated in part by activation of PKC, and that insulin may be a physiologic trigger of this activation.     

Seasonal variation of nitrogen transformations in the pelagial of selected nearshore waters of the Baltic Sea with emphasis on the particulate pool

Physical and chemical conditions, particulate matter and N-uptake were characterized at two sampling sites at the eastern German coast of the Baltic Sea (Pomeranian Bay) over the annual period of 1997 (February–November). The inshore sampling sites (5 m water depth) differed with respect to the potential influences of river run-off and salt water exchange (mean values of salinity: 7.05 and 8.72 PSU), respectively. The mean org-C_diss/org-C_part-ratios (4.9 and 12.6) fell in the same order of magnitude (1.0–12.6) as values of neighboring inshore waters, and increasing values reflect an enhancement of the trophic level. Beside differences of nitrogen concentrations (dissolved inorganic nitrogen: 1.8–23.8 and 0.9–9.9 mol l^–1), particulate nitrogen (4.30–41.01 and 2.69–9.08 mol l^–1) and absolute uptake of N-nutrients (mean sum of NH₄ ⁺, urea, NO₃ ^– uptake rates: 0.141 and 0.087 mol l^–1 h^–1), specific uptake of ¹⁵N-labelled nutrients (NH₄ ⁺, urea, NO₃ ^–) as well as the relationships between the measured variables characterize distinguishable inshore systems. The high variability at the shallow sampling sites prevents, however a simple resolution of the seasonal courses. Light dose could be identified as a potential key in order to describe long-term variations of N-uptake at the station with higher organic matter concentration (station KW), but phytoplankton development is better reflected in the seasonal course of N-uptake at the other station. Specific nitrogen uptake rates (NH₄ ⁺: 0.0009–0.0353 h^–1, urea: 0.0001–0.0137 h^–1, NO₃ ^–: 0.000004–0.0009 h^–1) and relative nitrogen preferences indicate extraordinary importance of reduced nitrogenous nutrients (NH₄ ⁺, urea) at both stations throughout the year.     

Characterization of MgATP-Driven H+ uptake in to a microsomal vesicle franction from rat pancreatic acinar cells

Summary In microsomal vesicles, as isolated from exocrine pancreas cells, MgATP-driven H⁺ transport was evaluated by measuring H⁺-dependent accumulation of acridine orange (AO). Active H⁺ uptake showed an absolute requirement for ATP with simple Michaelis-Menten kinetics (K _m for ATP 0.43 mmol/liter) with a Hill coefficient of 0.99. H⁺ transport was maximal at an external pH of 6.7, generating an intravesicular pH of 4.8. MgATP-dependent H⁺ accumulatioin was abolished by protonophores. such as nigericin (10^–6 mol/liter) or CCCP (10^–5 mol/liter), and by inhibitors of nonmitochondria H⁺ ATPase, such as NEM or NBD-Cl, at a concentration of 10^–5 mol/liter. Inhibitors of both mitochondrial and nonmitochondrial H⁺ pumps, such as DCCD (10^–5 mol/liter) or Dio 9 (0.25 mg/ml), reduced microsomal H⁺ transport by about 90%. Vanadate (2×10^–3 mol/liter). a blocker of those ATPases, which form a phosphorylated intermediate, did not inhibit H⁺ transport. The stilbene derivative DIDS (10^–4 mol/liter), which inhibits anion transport systems, abolished H⁺ transport completely. MgATP-dependent H⁺ transport was found to be anion dependernt in the sequence Cl^–>Br^–>gluconate^–; in the presence of SO ₄ ^–2 . CH₃COO^– or No ₃ ^– , no H⁺ transport was observed. MgATP-dependent H⁺ accumulation was also cation dependent in the sequence K⁺>Li⁺>Na⁺=choline⁺, As shown by dissipation experiments in the presence of different ion gradients and ionophores, both a Cl^– and a K⁺ conductance, as well as a small H⁺ conductance. were found in the microsomal membranes. When membranes containing the H⁺ pump wer further purified by Percoll gradient centrifugatin (ninefold enrichment comparad to homogenate), no correlation with markers for endoplasmic reticulum., mitochondria, plasma membranes, zymogen graules or Golgi membranes was found.The present data indicate that the H⁺ pump located in microsomes from rat exocrine pancreas is a vacuolar-or V-type H⁺ ATPase and has most similarities to that described in endoplasmic reticulum. Golgi apparatus or endosomes.     

On the cross-reactivity of amiloride and 2,4,6 Triaminopyrimidine (TAP) for the cellular entry and tight junctional cation permeation pathways in epithelia

Summary 2,4,6 Triaminopyrimidine (TAP) has been previously shown to inhibit the passive tight junctional cation permeation pathway in various leaky epithelia. Amiloride has been shown to be an effective inhibitor of the cation cellular entry pathway in tight epithelia. In this paper we demonstrate that TAP and amiloride at appropriate concentrations are able to block either of these epithelial cation permeation pathways. TAP was found to block the Na entry pathway in frog skin with the following characteristics: it (1) inhibits from the external solution only, (2) is completely reversible, (3) increases the transepithelial resistance, (4) is active in the monoprotonated form, (5) is noncompetitive with Na, (6) displays saturation kinetics which obey a simple kinetic model (K _I=1×10^–3 m), (7) is independent of external calcium, (8) is dependent on external buffering capacity, and (9) is competitive with amiloride. Amiloride inhibition of the junctional permeation in gallbladder had the following characteristics: it (1) increases the transepithelial resistance, (2) decreases cation conductance without affecting the anion conductance, (3) displays saturation kinetics which obey a simple kinetic model (K _I=1×10^–3 m), and (4) possesses inhibitory activity in both its protonated and unprotonated form. These results not only indicate that a similar inhibitory site may exist in both of these cation permeation pathways, but also provide information on the chemical nature and possible location of these inhibitory sites.     

Sodium influx in isolated gills of the freshwater mussel,Ligumia subrostrata

Summary Isolated gills of the freshwater mussel,Ligumia subrostrata, accumulate Na from a pondwater bathing medium. The rate of Na transport by the isolated gill is 13.2±1.1 mol (g dry gill·10 min)^–1 which equals or exceeds the estimated Na transport rate of intact animals. Sodium influx is saturable with aV _max of 13.6±1.2 mol (g dry gill·10 min)^–1 and an affinity (K _s) of 0.17 mM Na/l. The isolated gills survive prolonged exposure to pondwater with a constant of 890 l O₂ (g dry gill·h)^–1 over a 4 h period. Sodium transport in the isolated gills is stimulated 80% above control values by 10^–4 M serotonin, 60% by 0.5 mM cAMP and 60% by 12.5 g/ml nystatin. Sodium influx is inhibited by 0.5 mM amiloride and 1 mM lithium.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Measurement of apoplasmic and cell-to-cell components of root hydraulic conductance by a pressure-clamp technique

A pressure-clamp technique was devised for the direct measurement of cell-to-cell and apoplasmic components of root hydraulic conductance; the experimental results were analyzed in terms of a theoretical model of water and solute flow, based on a composite membrane model of the root. When water is forced under a constant pressure into a cut root system, an exponential decay of flow is observed, until a constant value is attained; when pressure is released, a reverse water flow out of the root system is observed which shows a similar exponential behavour. The model assumes that the transient flow occurs through a cell-to-cell pathway and the observed decrease is the result of accumulation of solutes in front of the root semi-permeable membrane, whilst the steady-state component results from the movement of water through the parallel apoplasmic pathway. Root conductance components are estimated by fitting the model to experimental data. The technique was applied to the root systems of potted cherry (Prunus avium L.) seedlings; average apoplasmic conductance was 15.5 × 10^–9m³· s^–1· MPa^–1, with values ranging from 12.0 × 10^–9 to 18.5 × 10^–9m³· s^–1· MPa^–1; average cell-to-cell conductance was 11.7 × 10⁹ m³· s^–1· MPa^–1, with values ranging from 8.5 × 10^–9 to 15.3 × 10^–9 m³ · s^–1·MPa^–1. Cell-to-cell conductance amounted on average to 43% of total root conductance, with values between 41 and 45%. Leaf specific conductance (conductance per unit of leaf area supported) of the root systems ranged from 2.7 × 10^–8 to 5.6 × 10^–8 m· s^–1·MPa^–1, with an average of 3.7 × 10^–8 m · s^–1·MPa^–1. The newly developed technique allows the interaction of mass flow of water and of solutes to be explored in the roots of soil-grown plants.Abbreviations and Symbols A L_p root hydraulic conductance - A^aL _p ^a root apoplasmic conductance - A^ccL _p ^cc root cell-to-cell conductance - C_s(t) concentration of solutes in apical root compartment at time t - J_v flow of water through the root - J _v ^a apoplasmic flow of water - J_v/cc cell-to-cell flow of water - LSC leaf specific conductance of the root system - P root hydrostatic pressure - P_appl applied pressure - _s(t) root osmotic pressure at time t - _m osmotic pressure of rooting medium - reflection coefficient of root membrane - time constant of cell-to-cell flow decay This research was funded within the EC Project Long-term effects of CO₂-increase and climate change on European forests (LTEEF) (EV5V-CT94-0468); F.M. was supported by a Ministero dell' Universitá e della Ricerca Scientifica e Tecnologica — British Council agreement (Project The ecological significance of cavitation in woody plants); M.C. was supported by a Consiglio Nazionale delle Ricerche — British Council agreement. We gratefully thank Prof. P.G. Jarvis (University of Edinburgh, UK) for revising an earlier version of this paper and Prof. E. Steudle (University of Bayreuth, Germany) for helpful comments.     

Analysing the responses of photosynthetic CO2 assimilation to long-term elevation of atmospheric CO2 concentration

Understanding how photosynthetic capacity acclimatises when plants are grown in an atmosphere of rising CO₂ concentrations will be vital to the development of mechanistic models of the response of plant productivity to global environmental change. A limitation to the study of acclimatisation is the small amount of material that may be destructively harvested from long-term studies of the effects of elevation of CO₂ concentration. Technological developments in the measurement of gas exchange, fluorescence and absorption spectroscopy, coupled with theoretical developments in the interpretation of measured values now allow detailed analyses of limitations to photosynthesisin vivo. The use of leaf chambers with Ulbricht integrating spheres allows separation of change in the maximum efficiency of energy transduction in the assimilation of CO₂ from changes in tissue absorptance. Analysis of the response of CO₂ assimilation to intercellular CO₂ concentration allows quantitative determination of the limitation imposed by stomata, carboxylation efficiency, and the rate of regeneration of ribulose 1:5 bisphosphate. Chlorophyll fluorescence provides a rapid method for detecting photoinhibition in heterogeneously illuminated leaves within canopies in the field. Modulated fluorescence and absorption spectroscopy allow parallel measurements of the efficiency of light utilisation in electron transport through photosystems I and IIin situ.Abbreviations A net rate of CO₂ uptke per unit leaf area (µmol m^–2 s^–1) - A_sat light-saturated A - A₈₂₀ change in absorptance of PSI on removal of illumination (OD) - c CO₂ concentration in air (µmol mol^–1) - c_a c in the bulk air; c_i, c in the intercellular spaces - ce carboxylation efficiency (mol m^–2 s^–1) - E transpiration per unit leaf area (mol m^–2 s^–1) - F fluorescence emission of PSII (relative units) - F_m maximal level of F - F_o minimal level of F upon illumination when PSII is maximally oxidised - F_s the steady-state F following the m peak - F_v the difference between F_m and F_o - F'_m maximal F' generated after the m peak by addition of a saturating light pulse - F'_o the minimal level of F' after the m peak determined by re-oxidising PSII by far-red light - g₁ leaf conductance to CO₂ diffusion in the gas phase (mol m^–2 s^–1) - g'₁ leaf conductance to water vapour diffusion in the gas phase (mol m^–2 s^–1) - k_c and k_o the Michaelis constants for CO₂ and O₂, respectively, (µmol mol^–1); - J_max the maximum rate of regeneration of rubP (µmol m^–2 s^–1) - l stomatal limitation to CO₂ uptake (dimensionless, 0–1) - LCP light compensation point of photosynthesis (µmol m^–2 s^–1) - o_i the intercellular O₂ concentration (mmol mol^–1) - Pi cytosol inorganic phosphate concentration - PSI photosystem I - PSII photosystem II - Q photon flux (µmol m^–2 s^–1) - Q_abs Q absorbed by the leaf - rubisCO ribulose 1:5 bisphosphate carboxylase/oxygenase; rubP, ribulose 1:5 bisphosphate; s, projected surface area of a leaf (m²) - V_c,max is the maximum rate of carboxylation (µmol m^–2 s^–1) - W_c the rubisCO limited rate of carboxylation (µmol m^–2 s¹) - W_j the electron transport limited rate of regeneration of rubP (µmol m^–2 s^–1) - W_p the inorganic phosphate limited rate of regeneration of rubP (µmol m^–2 s^–1) - absorptance of light (dimensionless, 0–1) - _a of standard black absorber ₁, of leaf - _s of integrating sphere walls - , CO₂ compensation point of photosynthesis (µmol mol^–1) - the specificity factor for rubisCO carboxylation (dimensionless) - , convexity of the response of A to Q (dimensionless 0–1) - the quantum yield of photosynthesis on an absorbed light basis (A/Q_abs; dimensionless) - the quantum yield of photosynthesis on an incident light basis (A/Q; dimensionless) - _app the maximum - _m the maximum - _m,app the photochemical efficiency of PSII (dimensionless, 0–1) - _PSII,m the maximum      

Technical communication: Determination of cuprous ions in bacterial leachates and for environmental monitoring

A sensitive and precise spectrophotometric method has been developed for the determination of copper(I) in bacterial leach liquors produced by the action of Thiobacillus ferrooxidans and T. thiooxidans on copper ores. In this method bicinchoninic acid (BCA) has been used as the chromogenic reagent which produces a stable purple complex with Cu(I) which was found to obey Beer's Law and with _max at 560 nm. The coloured complex has a molar extinction coefficient () value of 6.6 × 10³ l mol^–1 cm^–1; specific absorptivity () value of 0.104 ml^–1 g cm^–1 and the Sandell sensitivity (S) value was 0.0096 g cm². Optimal conditions for development of coloration/sensitivity were determined. Interferences due to cations and anions were investigated and various masking agents for alleviating their inhibition were studied. The method has been found very useful in determining ratios of Cu(I) to Cu(II) in bacterial leach liquors and should play a significant role in determining the reaction mechanisms of biological leaching and for environmental monitoring.     

Photosynthesis and Water Use Efficiency in Twenty Tropical Tree Species of Differing Succession Status in a Brazilian Reforestation

Leaf gas exchange characteristics were measured in twenty woody species that differ in succession status ranging from pioneer species (PS) to late succession species (LS) in a Brazilian rain-reforestation ecosystem. Photon-saturated photosynthetic rate, calculated per either a leaf area (P _NA) or a dry mass (P _NM) basis, differed among species. P _NA and P _NM were highest in PS and lowest in LS. Variation among species was 3-fold (from 7 to 23 mol m^–2 s^–1) for P _NA, and 5-fold (from 50 to 275 mol kg^–2 s^–1) for P _NM. The highest P _NA (23 mol m^–2 s^–1) and P _NM (275 mol kg^–2 s^–1) values were recorded in PS Croton urucurana, while the lowest P _NA (7 mol m^–2 s^–1) and P _NM (50 mol kg^–2 s^–1) values were recorded in LS Aspidosperma cylindrocarpon. A considerable overlap was recorded between PS and LS in values of stomatal conductance (g _s), transpiration rate (E), and leaf mass to area ratio (ALM). However, C. urucurana also showed highest g _s and E. P _NM was highly correlated with ALM in both PS and LS (r=–0.75 and –0.90, respectively). The high values of instantaneous transpiration efficiency (ITE) and intrinsic water use efficiency (WUE_i) were also observed in the PS when compared with the LS.     

Nutrient content and photosynthesis of young yellowing Norway spruce trees (Picea abies L. Karst.) following calcium and magnesium fertilisation

Severely yellowed ten-year-old spruce trees growing in the Vosges Mountains on an acidic soil were fertilised with Magnesium lime during the spring of 1990. The effects of this treatment were assessed 18 months later. A very significant improvement of the mineral status of the trees was detected, with increasing Mg contents in the needles, and as a consequence, reduced yellowing and improved chlorophyll content. Only slight differences with control trees were observed for height increase. Effects of this improved nutrition on photosynthesis were tested measuring net CO₂ assimilation rates and chlorophyll a fluorescence. Light-saturated net assimilation rates of current-year needles were high, reaching 5.3 mol m^–2 s^–1 on a total needle area basis. The improvement in chlorophyll and Mg content had no significant effect on net assimilation rates or on any parameter describing photochemical functions of both current-and previous-year needles. Despite the strong inter-individual variability in needle chlorophyll and Mg contents (ranging from 0.2 to 0.8 mg g^–1 fresh weight, and 0.05 to 0.5 mg g^-1 dry weight respectively), photochemical efficiency of PS II under limiting irradiance only decreased significantly on older needles displaying Mg contents below 0.1 mg g^–1. It is concluded from these results that spruce trees exhibit a high degree of plasticity with regard to Mg deficiency on acidic soils, and that improved Mg nutrition and increased chlorophyll content do not necessarily improve photosynthesis and height growth.Abbreviations A light-saturated net CO₂ assimilation rate (mol m^–2 s^–1) - g_w light-saturated needle conductance to water vapour (mmol m^–2 s^–1) - _wp and _wm pre-dawn and mid-day needle water potential (MPa) - osmotic potential of sap expressed from needles (MPa) - PFD photosynthetic photon flux density (mol m^–2 s^–1) - F_v/F_m photochemical efficiency of PS II after 20 min dark adaptation - F/F_m ^' photochemical efficiency of PS II reaction centres after 10 min at a PFD of 220 mol m^–2 s^–1     

A channel that allows inwardly directed fluxes of anions in protoplasts derived from wheat roots

An anion channel that only allows outward current flow (anion influx) has been identified in protoplasts derived from wheat (Triticum aestivum L., Triticum turgidum L.) roots. The anion outward rectifier (anion OR) measured by patch-clamp of whole cells activated very quickly, usually reaching a steady-state level in less than 100 ms and was easily distinguished from the cation outward rectifier (cation OR) which activated more slowly during membrane depolarisation. The anion OR is permeable to NO ₃ ^– and Cl^–, moderately permeable to I^–, and relatively impermeable to H₂PO₄/^– and ClO₄/^–. An anomalous mole-fraction effect between ClO_4/ ^– and Cl^– was observed on the outward current, indicating that the channel is a multi-ion pore. The anion OR is gated by both voltage and external anion concentration such that it activates near to the equilibrium potential for the permeant anion. It activated at more negative membrane potentials when NO ₃ ^– was substituted for Cl^– in the external medium, indicating that the channel may function to allow NO ₃ ^– influx under luxuriant external NO ₃ ^– concentrations. For most experiments, K⁺ and Cl^– were the main cation and anion in solution, and under these conditions it appeared likely that the anion OR functioned in membrane-potential regulation by facilitating a Cl^– influx at membrane potentials more positive than the chloride reversal potential (E_Cl). If E_Cl was more negative than the K⁺ reversal potential (E_K) then the anion OR dominated but both the anion and cation ORs occurred together when the membrane potential difference (V_m) was positive of both E_Cl and E_K. The cation OR was inhibited by increasing external Cl^– concentrations, but the anion OR was not affected by external K⁺ or Na⁺ concentration. The anion-transport inhibitors, zinc and phenylglyoxal were ineffective in blocking the anion OR. 4,4-Di-isothiocyanostilbene-2, 2-disulfonic acid (DIDS) irreversibly blocked about 34% of the current when applied extracellularly at a concentration of 25 M, and about 69% at a concentration of 200 M. However, DIDS (200 M) also occasionally acted as an irreversible blocker of the cation OR. Perchlorate blocked irreversibly 75% of the current at an external concentration of 10 mM and did not block the cation OR. Whole-cell currents also indicated that the anion OR was insensitive to external pH (pH=5–7) and calcium concentration ([Ca²⁺]=0.1–10 mM). Increasing intracellular calcium concentration significantly increased the occurrence of the fast outward current in whole cells (P < 0.005, X² test). With approximately 10 nM calcium inside the cell the anion outward current was observed in 64% (n = 45) of cells and with 50 nM calcium inside the cell the anion current was observed in 88% (n = 69) of cells. Single-anion OR channels observed in outside-out patches had a conductance in 300 mM KCl (external) of about 4 pS. When voltage pulses were applied to outside-out patches the average currents were similar to those observed in whole cells. The significance of the anion OR as a likely route for Cl uptake in high salinities is discussed.Abbreviations Bath solution bathing the extracellular face of the membrane - DIDS (4,4-diisothiocyanostilbene-2,2-disulfonic acid) - E_x reversal potential for ion x - OR outward rectifier - Pip solution inside the pipette - TEACl (tetraethyl-ammonium chloride) - V_m membrane potential difference We thank the Australian Research Council for financial support, G.P. Findlay and A. Garrill for helpful discussions, and K. Morris and D. Mackenzie for expert technical assistance. M.S. was supported by an Australian Postgraduate Research Award.     

Application of N-15 dilution for simultaneous estimation of nitrification and nitrate reduction in soil-water columns

Nitrification and denitrification rates were estimated simultaneously in soil-floodwater columns of a Crowley silt loam (Typic Albaqualfs) rice soil by an¹⁵N isotopic dilution technique. Labeled NO ₃ ^– was added to the floodwater of soil-water columns, half were treated with urea fertilizer. The (NO ₃ ^– +NO ₂ ^– )–N and (NO ₃ ^– +NO ₂ ^– )–N concentrations in the floodwater were measured over time and production and reduction rates for NO ₃ ^– calculated. Nitrate reduction in the urea amended columns averaged 515 mol N m^–2h^–1 and nitrification averaged 395 mol N m^–2h^–1 over the 35–153 d incubation. The nitrification rate for 4–19 d sampling period (1,560 mol N m^–2h^–1) in the urea amended columns was almost 9 times greater than the reduction rate (175 mol N m^–2h^–1) over the same period. Without the addition of urea the NO ₃ ^– production rate averaged 32 mol N m^–2h^–1 and reduction 101 mol N m^–2h^–1.     

Ferrochelatase activity inAzospirillum brasilense with reference to the influence of metal cations

Summary Ferrochelatase in membrane preparations fromAzospirillum brasilense displayed an activity of 2.17 mol protoheme formed · h^–1 · mg protein^–1 which is 10-fold greater than previous reports for other bacteria. This ferrochelatase showed an apparentK _m of 20.9 M for Fe²⁺, a pH optimum of 6.0–6.5, and stimulation by oleic or stearic acids. Co²⁺, Cu²⁺ and Zn²⁺ inhibited the incorporation of Fe²⁺ into protoporphyrin IX while Ni² and Mg²⁺ had no effect on protoheme synthesis. Activity with Fe²⁺ and mesoporphyrin IX was less than with protoporphyrin IX but deuteroporphyrin IX produced the highest rate of protoheme synthesis. The membrane fraction containing ferrochelatase activity was found to insert Cu²⁺, Ni²⁺, Zn²⁺ and Co²⁺ enzymatically into protoporphyrin IX to produce metalloporphyrins. Cu²⁺ incorporation into protoporphyrin IX proceeded at a rate greater than with Fe²⁺ and theK _m for Cu²⁺ was 21.9 M.     

Presence of an X AG − carrier in frog (Rana esculenta) red blood cells

Evidence is presented that the high levels of internal l-glutamic and l-aspartic acid in frog Rana esculenta red blood cells are due to the existence of a specific carrier for acidic amino acids of high affinity K _m = 3 m and low capacity (V_max) 0.4 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1. It is Na+ dependent and the incorporation of l-glutamic acid can be inhibited by l and d-aspartate and l-cysteic acid, while d-glutamic does not inhibit. Moreover, this glutamic uptake shows a bell-shaped dependence on the external pH. All these properties show that this carrier belongs to the system X _AG ^– family. Besides the incorporation through this system, l-glutamic acid is also taken up through the ASC system, although, under physiological conditions, this transport is far less important, since it has relatively low affinity K _m 39 m but high capacity (V _max) 1.8 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1.     

Enzymatic transformation of desacetyl-lanatoside A into digitoxin by β-glucosidase action in cyclodextrin solutions

Summary The enzymatic transformation of desacetyl-lanatoside A (DLA) to its secondary glycoside, digitoxin, in solutions of -and -cyclodextrins is effected using of -glucosidase from barley. Due to the interaction of cyclodextrins (CyDs) with desacetyl-lanatoside A, an increase in solubility of the latter of 24.5 and 230 times was observed for -cyclodextrin and -cyclodextrin, respectively. Kinetic studies of the enzymatic transformation gave for -glucosidase the values K_M=3.3×10^–4 mol. dm^–3 and V_max=0.557 mol mg^–1 min^–1 when the substrate was the deacetyl-lanatoside A complex with -cyclodextrin, while in the case of the complex with -cyclodextrin these values were K_M=5.45×10^–4 mol dm^–3 and V_max=0.896 mol mg^–1 min^–1.     

Intercellular CO2 concentration and water-use efficiency of temperate plants with different life-forms and from different microhabitats

Summary Photosynthesis and transpiration were measured simultaneously, under near-optimum and constant environmental conditions, in intact leaves of plants native to the temperate forest region. A linear relationship between photosynthetic rate and stomatal conductance was found in every species tested irrespective of leaf age or season, indicating that the calculated intercellular CO₂ concentration and water-use efficiency were fairly constant within a species. The values of intercellular CO₂ concentration and water-use efficiency ranged from 221 to 271 l l^–1 and 4.46 to 8.20 mol CO₂ mmolH₂O^–1 (6.24±0.90 mol CO₂ mmolH₂O^–1), respectively. The variations in intercellular CO₂ concentration and water-use efficiency were not directly related to photosynthetic capacities, life-forms, or microhabitat preferences. The intercellular CO₂ concentrations found in this study were close to values reported from cultivated plants and plants native to more arid regions, suggesting a common mechanism to maintain the stomatal conductance proportional to photosynthetic capacity over a wide variety of C₃ plants.     

ATP formation coupled to caffeate reduction by H2 in Acetobacterium woodii NZva16

We have addressed the question, whether the reduction of caffeate in Acetobacterium woodii strain NZva16 is coupled to ATP synthesis by electron transport phosphorylation. The following results were obtained: 1. Cultures of A. woodii with H₂ and CO₂, grew to greater cell densities, when caffeate was also present. Caffeate was reduced to give hydrocaffeate and less acetate was formed. The cell yield based on the amount of caffeate reduced was approximately 1 g dry cells/mol. 2. Non-growing bacterial suspensions catalyzed the reduction of caffeate by H₂. The specific activity (0.2–1.0 mol · min^–1 · mg^–1 bacterial protein) was as high as expected for a catabolic reaction. 3. The ATP content of bacteria incubated, with H₂ increased from < 1 to about 7 mol per g cellular protein on the addition of caffeate. The ATP yield was calculated as 0.06 mol ATP · mol^–1 caffeate from the initial velocity of ATP formation and the activity of caffeate reduction. Valinomycin together with nigericin inhibited ATP formation and caused a 2–3-fold increase of the activity of caffeate reduction. Protonophores were without, effect. 4. Caffeate in the presence of H₂ caused the uptake of tetraphenylphosphonium cation by the bacteria. The uptake was abolished by valinomycin plus nigericin, and was considerably enhanced by monensin. Protonophores were without effect, even in the presence of monensin. It is concluded that caffeate reduction by H₂ is coupled to ATP formation by electron transport phosphorylation. However, the failure of protonophores to prevent phosphorylation and TPP uptake cannot be explained.Abbreviations Caffeate 3,4-Dihydroxycinnamate - Hydrocaffeate 3,4-dihydroxyphenylpropionate - TPP⁺ tetraphenylphosphonium cation - FCCP carbonylcyanide-4-trifluoromethoxyphenylhydrazone - TTGB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazol - TCS 3,5,3,4-tetrachlorosalicylanilide