Uptake of Thallium, a Toxic Heavy-Metal, in the Cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. Leopoliensis) PCC 7942

Uptake of the toxic heavy-metal, thallium, was studied in thecyanobacterium Synechococcus R-2 (PCC 7942) using clinicallyavailable ²⁰¹Tl ⁺. Thallium was found to distribute across theplasmalemma passively, and so the accumulation ratio of theion ([Tl⁺]_i/[Tl⁺]_o) could be used to calculate the apparentmembrane potential (­_i,o) of the cells (^ETI⁺_i,o = ­_i,o).The permeability of the plasmalemma to TI⁺ (^PTI⁺ 1 to 5 nms^–1)is higher than that of K⁺. Valinomycin does not increase thepermeability of TI⁺. Transient changes in the ­_i,o of cells,because of electrogenic transport of ions, could be detectedfrom its effects upon the uptake rate of TI⁺. HCO^–₃ hyperpolarizedSynechococcus cells, whereas NH⁺₄, CH₃NH⁺, and K⁺ led to depolarization.The use of TI⁺ as a reporter of ­_i,o has some inherent limitations.Tl⁺ is toxic at very low concentrations (inhibitory effectsare apparent after about 6 h at concentrations as low as 1 mmolm^–3). The rate of equilibration is slow (t_1/25 to 20 min).Equilibration of TI⁺ takes about 2 h, which limits its valueas a membrane potential probe. Large amounts of TI⁺ bind tothe surface of the cells making the method impracticable formeasuring accumulation ratios of less than about 10 (­_i,o)values smaller than about –60 mV). Cultures continuouslyexposed to Tl⁺ (10 mmol m^–3) eventually become TI⁺ resistantby actively extruding TI⁺ (µTI⁺_i,o= –3±0.2kJ mol^–1) and so thallium cannot be used as a ­_i,oprobe in such cells. (Received October 28, 1997; Accepted August 31, 1998)     

A comparison of the impacts of various nitrogen sources on acid-base balance in C3 Triticum aestivum L. and C4 Zea mays L. plants

This work aimed to study the impacts of acquisition and assimilationof various nitrogen sources, i.e. NO₃^–, NH₄⁺ or NH₄NO₃,in combination with gaseous NH₃ on plant growth and acid-basebalance in higher plants. Plants of C₃ Triticum aestivum L.and C₄ Zea mays L. grown with shoots in ambient air in hydroponicculture solutions with 2 mol m^–3 of nitrogen source asNO₃^–, NH₄⁺ or NH₄NO₃ for 21 d and 18 d, respectively,had their shoots exposed either to 320 µg m^–3 NH₃or to ambient air for 7 d. Variations in plant growth (leaves,stubble and roots), and OH^– and H⁺ extrusions as wellas the relative increases in nitrogen, carbon and carboxylatewere determined. These data were computed as H⁺/N, H⁺/C, (C-A)/N,and (C-A)/C to analyse influences of different nitrogen sourceson acid-base balance in C₃ Triticum aestivum and C₄ Zea maysplants. Root growth in dry weight gain was significantly reduced bytreatment with 320 µg m^–3 NH₃ in Triticum aestivumand Zea mays growing with different N-forms, whereas leaf growthwas not significantly affected by NH₃. In comparison with C₃Triticum aestivum, non-fumigated C₄ Zea mays had low ratiosof OH^–/N in NO₃^–3-grown plants and of H⁺/N in NH₄⁺- and NH ₄NO₃-grown plants. Utilization of NH₃ from the atmospherereduced both the OH^–N ratios in NO₃^– -grown plantsand the H⁺/N ratio in NH₄⁺ - and NH₄NO₃ -grown plants of bothspecies. Furthermore, Zea mays had higher ratios of (C-A)/Nin NH₄⁺ - and NH₄NO₃-grown plants than Triticum aestivum. Thismeans that C₄ Zea mays had synthesized more organic anion perunit increase in organic N than C₃ Triticum aestivum plants.Within both species, different nitrogen sources altered theratios of (C-A)/N in the order: NH₄NO₃>NH₄⁺>NO₃^–.Fumigation with NH₃ increased organic acid synthesis in NO₃^–- and NH₄⁺ - grown plants of Triticum aestivum, whereas it decreasedorganic acid synthesis in Zea mays plants under the same conditions.Furthermore, these differences in acid-base regulation betweenC₃ Triticum aestivum and C₄ Zea mays plants growing with differentnitrogen sources are discussed. Key words: Acid-base balance, ammonia, ammonium, nitrate, ammonium nitrate, C₃ Triticum aestivum L., C₄ Zea mays L.     

Effects of algal concentration, bacterial size and water chemistry on the ingestion of natural bacteria by cladocerans

Journal of Plankton Research, 11, 1273–1295, 1989. The values of P/U⁰ (Table I) and fluid velocity used to calculatethe energy required for sieving (pp. 1289–1290) and severalequations (footnote b of Table I; p. 1290, lines 3–4)are incorrect. The corrected table appears below: Table I. Filter setule measurements (mean and within specimenstandard deviation) of the gnathobases for the cladocerans studied^aGnathobaseof trunklimb number. ^bP = 8µU₀/(b(1 – 21nt + 1/6(t²) - 1/144(t⁴))), whereP = pressure drop in dyn cm^–2, =3.1416, U₀ = fluid velocityin cm s^–1, b = distance between setule centres in cm,t = ( x setule diameter)/b and µ = 0.0101 dyn s^–1cm^–2. Formula from Jørgensen (1983). The text (p. 1289, line 19 to p. 1290, line 10) should read: organism. Using a similar argument, a 0.5 mm Ceriodaphnia witha filter area of 0.025 mm² (Ganf and Shiel, 1985) and pressuredrop P = 2757 dyn cm^–2 (with fluid velocity of 0.07 cms^–1) allocates only 2171 ergs h^–1 to filtrationof a total energy expenditure of 10⁴ ergs h^–1 [filtrationenergy (ergs h^–1) = area (cm²) x pressure drop (dyn cm^–2)x 3600 (s h^–1) x 1/0.2 (efficiency of conversion of biochemicalinto mechanical work); total energy (ergs h^–1) = respiration(0.05 µl O₂ ind^–1 h^–1 consumed; Gophen, 1976)x conversion factor (2 x 10⁵ ergs µl^–1 O₂). Withan estimated 0.034 mm² in filter area, fluid velocity of 0.041cm s^–1 and respiration of 1.8 x 10⁴ ergs h^–1 (calculatedfrom Porter and McDonough, 1984), a 0.5 mm Bosmina uses <4%of its metabolism to overcome filter resistance. The velocities used in the original examples (0.4 cm s^–1for Ceriodaphnia, 0.2 cm s^–1 for Bosmina) were derivedfrom literature values of appendage beat rate and estimatesof the distance travelled by the appendages during each beatcycle. This approach unnecessarily assumes that all water movedpasses through the filter. In the new calculations, the flowacross the filter needed for food to be collected by sieving(0.07 cm s^–1 for Ceriodaphnia and 0.041 cm s^–1 forBosmina) was determined from the maximum clearance rate/filterarea. The amended energy expenditures, although higher, do notrefute the sieve model of particle collection.     

Phosphate and Membrane Electropotentials in Trifolium repens L.

In Trifolium repens L. there were immediate transient depolarizationsof the membrane electropotential (E_vo) when KH₂PO₄ was addedto phosphate-free media, but these were of the same magnitudeas the controls (K²SO⁴ and KCI). Furthermore, the extents ofdepolarization were the same as the expected effect of the addedK⁺ calculated using the Goldman equation. There was no significantdepolarization on adding H₃PO₄ to buffered media. Consequently,there was no evidence for a depolarization caused by phosphate.This result provides evidence that the H⁺–H₂PO₄ symportin roots of T. repens operates with a stoichiometry of 1: 1. In a group of control plants ( + P plants) and a group whichwere stressed by reducing the supply of phosphate (– Pplants), the – P plants had lower values for E_vo than+P plants (– 118 mV and – 130 mV, respectively).The absence of phosphate from the measurement media also reducedE_vo (mean effect = 9 mV). A significant difference in E_vo between– P and + P plants persisted when phosphate was addedto – P plants. The electropotential difference acrossthe tonoplast (E_vo) in – P plants became more positivewith time. Key words: White clover, membrane transport, roots, tonoplast, symport     

Intracellular pH Regulation in the Giant-Celled Marine Alga Chaetomorpha darwinii

The vacuolar pH (pH_v) and the cytoplasmic pH (pH_c) of the marinegiant-celled green alga Chaetomorpha darwinii were measuredby pH microelectrode techniques on extracted vacuolar sap, andby the [^I4C]DMO distribution method respectively. Equilibrationof DMO occurred with a half-time of about 2 h, with an apparentP_DMO of 3.6 x 10^–5 cm s^–1, but the vacuolar concentrationof free, undissociated DMO was always less than the externalconcentration. The explanation offered for freshwater giant-celledalgae of net DMO^– leakage across the plasmalemma cannotapply to Chaetomorpha darwinii, since electrically-driven DMO^–exit from the cytoplasm should be similar across the plasmalemmaand the tonoplast in these cells with large, vacuole-positivepotential differences across the tonoplast. pHc was accordinglycomputed assuming either tonoplast or plasmalemma equilibrationof DMO, with correction for DMO metabolism within the cell.pH_c was 8.0–8.3 in the light in artificial seawater (pH_oabout 8.0), was some 0.5 units lower in the dark, and was slightlylower with an external pH of 7. Vacuolar pH was 6.5–6.9,without consistent effects of illumination or of external pHof 7 rather than 8. While µ_H+ at the tonoplast was similarto that in giant-celled freshwater algae (although with a greatercontribution from relative to pH), µ_H+ at the plasmalemmawas less than 8 kJ mol^–1, i.e. less than one-third ofthe value in freshwater green algae. µ_Na+ was some 13kJ mol^–1 at the plasmalemma. The possibility that theprimary active transport process at the plasmalemma of Chaetomorphadarwinii (and certain other marine algae) is Na⁺ efflux ratherthan H⁺ efflux is discussed.     

Mitochondrial Ca2+-induced K+ influx increases respiration and enhances ROS production while maintaining membrane potential

We recently demonstrated a role for altered mitochondrial bioenergetics and reactive oxygen species (ROS) production in mitochondrial Ca²⁺-sensitive K⁺ (mtK_Ca) channel opening-induced preconditioning in isolated hearts. However, the underlying mitochondrial mechanism by which mtK_Ca channel opening causes ROS production to trigger preconditioning is unknown. We hypothesized that submaximal mitochondrial K⁺ influx causes ROS production as a result of enhanced electron flow at a fully charged membrane potential (_m). To test this hypothesis, we measured effects of NS-1619, a putative mtK_Ca channel opener, and valinomycin, a K⁺ ionophore, on mitochondrial respiration, _m, and ROS generation in guinea pig heart mitochondria. NS-1619 (30 µM) increased state 2 and 4 respiration by 5.2 ± 0.9 and 7.3 ± 0.9 nmol O₂·min^–1·mg protein^–1, respectively, with the NADH-linked substrate pyruvate and by 7.5 ± 1.4 and 11.6 ± 2.9 nmol O₂·min^–1·mg protein^–1, respectively, with the FADH₂-linked substrate succinate (+ rotenone); these effects were abolished by the mtK_Ca channel blocker paxilline. _m was not decreased by 10–30 µM NS-1619 with either substrate, but H₂O₂ release was increased by 44.8% (65.9 ± 2.7% by 30 µM NS-1619 vs. 21.1 ± 3.8% for time controls) with succinate + rotenone. In contrast, NS-1619 did not increase H₂O₂ release with pyruvate. Similar results were found for lower concentrations of valinomycin. The increase in ROS production in succinate + rotenone-supported mitochondria resulted from a fully maintained _m, despite increased respiration, a condition that is capable of allowing increased electron leak. We propose that mild matrix K⁺ influx during states 2 and 4 increases mitochondrial respiration while maintaining _m; this allows singlet electron uptake by O₂ and ROS generation. mitochondrial bioenergetics; heart mitochondria     

Corrigendum

Light response curves for (•) gross ¹⁶O₂ evolution, and() CO² uptake in 210 mmol mol^–1 O₂ with 900–1000µbar CO₂ or () in air by leaves of Hirschfeldia incana.The difference between (•) and () or () was quantitativelyequivalent to the measured ¹⁸O₂ uptake. The areas under thecurves are labelled to identify regions of assimilatory andnon-assimilatory electron flow redrawn from data of Canvin etal. (1980). It should be noted that the data and the labelling of the figureaxes are correct as printed.     

pH and Temperature Dependence of Tyrosine Z Decay Kinetics in Tris-Treated PSII Particles Studied by Time-Resolved EPR

The decay kinetics of tyrosine Z (Y_z) in Tris-treated PhotosystemII particles were measured by time-resolved EPR at differentpH values (pH 5.5 to 7.5) between 230 and 297 K. Y_z inducedby laser flashes decayed in a biphasic wav; t values were about100 ms in the fast phase and about 1 s in the slow one, respectively,at room temperature. The fast phase was attributed to a recombinationof charges on Y_z and Q^–_A. The activation energies forthe reaction of Y_z with Q^–_A between 245 and 297 K havebeen estimated to be more than 38 kJ mol^–1 at pH (>6.5)and less than 32 kJ mol^–1 at pH (<6.0), respectively.The activation energy gap between high pH (>6.5) and lowpH (<6.0) ranges was found to be E=4.4 kJ mol^–1. Judgingfrom the fact that pH 6.2 appears to be the border between thehigh and low pH regions, we suggest that the kinetics of Y_zis strongly influenced by the dissociation of the nearby histidineresidue. (Received February 21, 1997; Accepted June 24, 1997)     

Effect of Temperature and External pH on the Cytoplasmic pH of Chara corallina

Cytoplasmic pH (pH_c) in Chara corallina was measured (from [¹⁴C]stribution)as a function of external pH (pH₀)and temperature. With pH₀near 7, pH_c at 25?C is 7.80; pH_cincreases by 0.005 pH units?C^–1 temperature decrease, i.e. pH_c at 5 ?C is 7.90. WithpH? near 5.5, the increase in pH_c with decreasing temperatureis 0.015 units ?C^–1 between 25 and 15?C, but 0.005 units?C^–1 between 15 and 5?C. This implies a more precise regulationof pH_c with variations in pH_o at 5 or 15 ?C compared with 25?C. The observed dp H_c/dT is generally smaller than the –0.017units ?C^–1 needed to maintain a constant H⁺/OH^–1,or a constant fractional ionization of histidine in protein,with variation in temperature. It is closer to that needed tomaintain the fractional ionization of phosphorylated compoundsor of CO₂–HCO₃^– The value of dpH_c/dT has importantimplications for several regulatory aspects of cell metabolism.These include (all as a function of temperature) the rates ofenzyme reactions, the _H+ at the plasmalemma(and hence the energy available for cotransport processes),and the mechanism for pH_c regulation by the control of bidirectionalH⁺ fluxes at the plasmalemma.     

An Analysis of Some Effects of Humidity on Photosynthesis by a Tomato Canopy under Winter Light Conditions and a Range of Carbon Dioxide Concentrations

The rates of net photosynthesis by closed canopies of tomatoplants were measured at three CO₂ concentrations and three humiditiesover a range of natural light flux densities. The data havebeen analysed using a model of canopy photosynthesis which allowsfor variation in leaf area index and other leaf and canopy characteristics.The model also deals explicitly with the effects of CO₂ concentration,leaf conductance, and photorespiration on the leaf photochemicalefficiency, . The leaves were found to have a photochemicalefficiency in the absence of photorespiration, _m, of 12?6 ?10^–9 kg (CO₂) J^–1. At a CO₂ concentration of 0?73 ? 10^–3 kg m^–3 (400vpm) the leaf photochemical efficiency, , and canopy light utilizationefficiency, _c, were 18 per cent greater at a vapour pressuredeficit of 0?5 kPa than at 1?0 kPa. At a CO₂ concentration of2?2 ? 10^–3 kg m^–3 (1200 vpm) they were only 5 percent greater.     

Phosphate Uptake in the Cyanobacterium Synechococcus R-2 PCC 7942

Phosphate uptake rates in Synechococcus R-2 in BG-11 media (anitrate-based medium, not phosphate limited) were measured usingcells grown semi-continuously and in continuous culture. Netuptake of phosphate is proportional to external concentration.Growing cells at pH_o 10 have a net uptake rate of about 600pmol m^–2 s^–1 phosphate, but the isotopic flux for³²P phosphate was about 4 nmol m^–2 s^–1. There appearsto be a constitutive over-capacity for phosphate uptake. TheK_m and V_max, of the saturable component were not significantlydifferent at pH_o 7.5 and 10, hence the transport system probablyrecognizes both H₂PO^–₄and HPO^2–₄. The intracellularinorganic phosphate concentration is about 3 to 10 mol m^–3,but there is an intracellular polyphosphate store of about 400mol m^–3. Intracellular inorganic phosphate is 25 to 50kJ mol^–1 from electrochemical equilibrium in both thelight and dark and at pH_o 7.5 and 10. Phosphate uptake is veryslow in the dark ( 100 pmol m^–2 s^–1) and is light-activated(pH_o 7.51.3 nmol m^–2 s^–1, pH_o 10600 pmol m^–2s^–1). Uptake has an irreversible requirement for Mg²⁺in the medium. Uptake in the light is strongly Na⁺-dependent.Phosphate uptake was negatively electrogenic (net negative chargetaken up when transporting phosphate) at pH_o 7.5, but positivelyelectrogenic at pH_o 10. This seems to exclude a sodium motiveforce driven mechanism. An ATP-driven phosphate uptake mechanismneeds to have a stoichiometry of one phosphate taken up perATP (1 PO_{4 in}/ATP) to be thermodynamically possible under allthe conditions tested in the present study. (Received June 16, 1997; Accepted September 4, 1997)     

Measurement of Cytoplasmic pH by the DMO Technique in Hydrodictyon africanum

The 5, 5-dimethyl-[2-¹⁴C]oxazolidine-2, 4-dione (DMO) distributiontechnique for the measurement of intracellular pH has been appliedto giant cells of Hydrodictyon africanum. Significant metabolism of DMO was found in this alga; the free[DMO + DMO–] in subcellular samples is thus derived fromthe total label in cells equilibrated in [¹⁴C]DMO solutionsby measuring and subtracting the label in metabolic productsof DMO. A further problem arises from the observation that theDMO concentration in the vacuolar sap is always lower than thatpredicted by the transmembrane equilibration of undissociatedDMO from the bathing medium. This is interpreted in terms ofa finite permeability to the anion DMO–. Since the effectof P_DMO– on the DMO distribution is much smaller at thetonoplast (where the transmembrane electrical potential differenceis small) than at the plasmalemma, the values of cytoplasmicpH are computed assuming equilibration of undissociated DMOacross the tonoplast. At an external pH of 7.0 the cytoplasmic pH is about 7.4; decreaseor increase of external pH by 1 unit causes a decrease and anincrease in cytoplasmic p11 respectively of about 0.2 pH units.Determinations of _vo at pH 6, 7, and 8, together with an assumedconstant value of _cv, permit calculations of µ_H+ at theplasmalemma and tonoplast. The values are relatively independentof external pH in the range pH 6–8 at 21–25 and12–14 kJ mol^–1 respectively. The significance ofthese results for the regulation of intracellular pH, and forthe regulation and energising of the fluxes of ions, is discussed.     

C3 and CAM Photosynthetic Characteristics of the Submerged Aquatic Macrophyte Littorella uniflora: Regulation of Leaf Internal CO2 Supply in Response to Variation in Rooting Substrate Inorganic Carbon Concentration

The relationships between CO₂ concentrating mechanisms, photosyntheticefficiency and inorganic carbon supply have been investigatedfor the aquatic macrophyte Littorella uniflora. Plants wereobtained from Esthwaite Water or a local reservoir, with thelatter plants transplanted into a range of sediment types toalter CO₂ supply around the roots. Free CO₂ in sediment-interstitial-waterranged from 1–01 mol m^–3 (Esthwaite), 0.79 mol m^–3(peat), 0.32 mol m^–3 (silt) and 0–17 mol m^–3(sand), with plants maintained under PAR of 40 µmol m^–2s^–1. A comparison of gross morphology of plants maintained underthese conditions showed that the peat-grown plants with highsediment CO₂ had larger leaf fresh weight (0–69 g) andtotal surface area (223 cm² g^–1 fr. wt. including lacunalsurface area) than the sand-grown plants (0.21 g and 196 cm²g^–1 fr. wt. respectively). Root fresh weights were similarfor all treatments. In contrast, leaf internal CO₂ concentration[CO₂], was highest in the sand-grown plants (2–69 molm^–3, corresponding to 6.5% CO₂ in air) and lowest inthe Esthwaite plants (1–08 mol m^–3). Expressionof CAM in transplants was also greatest in the low CO₂ regime,with H⁺ (measured as dawn-dusk titratable acidity) of 50µmolg^– fr. wt., similar to Esthwaite plants in natural sediment.Assuming typical CAM stoichiometry, decarboxylation of malatecould account largely for the measured [CO₂]₁ and would makea major contribution to daytime CO₂ fixation in vivo. A range of leaf sections (0–2, 1–0, 5–0 and17–0 mm) was used to evaluate diffusion limitation andto select a suitable size for comparative studies of photosyntheticO₂ evolution. The longer leaf sections (17.0 mm), which weresealed and included the leaf tip, were diffusion-limited witha linear response to incremental addition of CO₂ and 1–0mol m^–3 exogenous CO₂ was required to saturate photosynthesis.Shorter leaf sections were less diffusion-limited, with thegreatest photosynthetic capacity (36 µmol O₂ g^–1 fr. wt. h^–1) obtainedfrom the 1.0 mm size and were not infiltrated by the incubatingmedium. Comparative studies with 1.0 mm sections from plants grown inthe different sediment types revealed that the photosyntheticcapacity of the sand-grown plants was greatest (45 µmolO₂ g^–1 fr. wt. h^–1) with a K_0.5 of 80 mmol m^–3.In terms of light response, saturation of photosynthesis intissue slices occurred at 850–1000 µmol m^–2s^–1 although light compensation points (6–11 µmolm^–2s^–1) and chlorophyll a: b ratios (1.3) were low.While CO₂ and PAR responses were obtained using varying numbersof sections with a constant fresh weight, the relationshipsbetween photosynthetic capacity and CO₂ supply or PAR were maintainedwhen the data were expressed on a chlorophyll basis. It is concludedthat under low PAR, CO₂ concentrating mechanisms interact inintact plants to maintain saturating CO₂ levels within leaflacunae, although the responses of the various components ofCO₂ supply to PAR require further investigation. Key words: Key words-Uttorella uniflora, internal CO₂ concentration, crassulacean acid metabolism, root inorganic carbon supply, CO₂ concentrating mechanism     

The Osmotic Pressure of Concentrated Solutions of Polyethylene Glycol 6000, and its Variation with Temperature

The vapour pressures of aqueous solutions of polyethylene glycol6000 have been measured (by equilibration with sucrose solutions)up to the saturation point at 25 °C (1.45 g g^–1 water).The reduced-osmotic-pressure (/c), when plotted versus concentration(c), rapidly and linearly increased up to a concentration ofabout 0.8 g g^–1 (crossing the similar plot for sucrose).Above this concentration, the reduced-osmotic-pressure rosemore slowly, but still more rapidly than sucrose. The maximumosmotic pressure achieved at saturation was nearly 18 MPa. Usingthe virial equation: /c= RT/M + RTA₂c, the calculated secondvirial coefficient (A₂) for the linear part is 4.5 x 10^–3mol g^–1, a value slightly greater than most literaturevalues at 25 °C. Data are cited showing that A₂ varies linearlyfrom 5–6 x 10^x3 at 0 °C, to zero at 80–90 °C     

Relation between Ultra-Violet Spectral Change and Nucleotide Binding of the Chloroplast Coupling Factor 1

Properties of the nucleotide binding sites on chloroplast couplingfactor 1 (CF₁) were studied by equilibrium dialysis and UV spectroscopy.From our direct binding studies, we identified at least fourkinds of ADP binding sites on CF₁; a barely dissociable ADPbinding site (site A), a slowly exchangeable high affinity sitewith dissociation constant (Kd) 0.021 µM (site B), anotherslowly exchangeable high affinity site with Kd 1.6 µM(site C) and several low affinity (Kd {small tilde}30 µM)sites. The Kd values for sites B and C of the other nucleotidestested were 0.5 µM and 16 µM (GDP), 8 µM and34 µM (CDP), 17 µM and 20 µM (UDP) and 1.4µM and 1.4 µM (PP₁). From a comparison of the observed UV spectral change and theamount of nucleotide bound to these sites, as calculated fromthe above Kd values, we concluded that the nucleotide bindingto site B or G induces UV spectral changes that are almost thesame in shape and magnitude. The estimated difference molarabsorption coefficient () was 3.4?10³M^–1_ADP cm^–1for ADP at 278 nm. Our conclusions were strengthened by thegood agreement between the observed spectra and the calculatedspectra (derived from the Kd and values of ADP and GDP) whenADP and GDP were added together to CF₁. The cause of the unusual behavior of GDP in the UV differencespectrum which was unexplained in our previous report was shownto be competition between the GDP added and previously boundADP at sites B and C; this distorted the real spectrum inducedby GDP. (Received October 3, 1983; Accepted February 13, 1984)     

Transnodal Transport of 14C in Nitella flexilis: II. TANDEM CELLS WITH APPLIED PRESSURE GRADIENTS

Using carefully standardized test conditions and tandem pairsof cells of Nitella flexilis, the influx of ¹⁴C (added as H¹⁴CO₃^–and transnodal transport were studied under various pressuregradients applied across the node up to P=± 2·5bar. When mannitol was used as the osmoticum, influx was foundto increase only when the mannitol solution was around the cellproximal to the feed. ¹⁴C was transported across the node tothe distal cell, probably as ¹⁴C-photosynthate products, evenagainst a pressure gradient of 2 bars or more, as was ³⁶Cl^–and ³²P. The % transported in general decreased with increasingP, whether assisted or opposed by added pressure. It was unchangedby the presence of mannitol at the node and was essentiallythe same whether the pressure gradient was produced by directpressure or by use of the osmoticum. Transnodal transport of¹⁴C products is almost certainly via plasmodesmata and appearsto be largely by an active mechanism. In absolute amounts itis the same whether the pressure gradient assists or opposesflow. Valving is evident at the node, increasing the resistanceto transport as the pressure gradient increases whether ¹⁴C(asHCO₃^–), ⁴²K⁺ (as KCl), ³⁶Cl^– (as NaCl) or ³²P (asNa₃PO₄) are used to detect it. The mechanism of movement ofK⁺ across the node differs from that of photosynthate products. Key words: Node, plasmodesmata, pressure gradients, active transport     

Estradiol-17beta stimulates proliferation of mouse embryonic stem cells: involvement of MAPKs and CDKs as well as protooncogenes

Although the importance of estradiol-17 (E₂) in many physiological processes has been reported, to date no researchers have investigated the effects of E₂ on embryonic stem (ES) cell proliferation. Therefore, in the present study, we have examined the effect of E₂ on the DNA synthesis of murine ES (ES-E14TG2a) cells and its related signaling pathways. The results of this study show that E₂ (10^–9 M) significantly increased [³H]thymidine incorporation at >4 h and that E₂ (>10^–12 M) induced an increase of [³H]thymidine incorporation after 8-h incubation. Moreover, E₂ (>10^–12 M) also increased 5'-bromo-2'-deoxyuridine (BrdU) incorporation and cell number. Indeed, E₂ stimulated estrogen receptor (ER)- and - protein levels and increased mRNA expression levels of protooncogenes (c-fos, c-jun, and c-myc). Tamoxifen (antiestrogen) completely inhibited E₂-induced increases in [³H]thymidine incorporation. In addition, estradiol-6-O-carboxymethyl oxime-BSA (E₂-BSA; 10^–9 M) increased [³H]thymidine incorporation at >1 h, and E₂-BSA (>10^–12 M) increased [³H]thymidine incorporation after 1-h incubation. E₂-BSA-induced increase in BrdU incorporation also occurred in a dose-dependent manner. Tamoxifen had no effect on E₂-BSA-induced increase of [³H]thymidine incorporation. Also, E₂ and E₂-BSA displayed maximal phosphorylation of p44/42 MAPKs at 10 and 5 min, respectively. E₂ increased cyclins D1 and E as well as cyclin-dependent kinase (CDK)2 and CDK4. In contrast, E₂ decreased the levels of p21^cip1 and p27^kip1 (CDK-inhibitory proteins). Increases of these cell cycle regulators were blocked by 10^–5 M PD-98059 (MEK inhibitor). Moreover, E₂-induced increase of [³H]thymidine incorporation was inhibited by PD-98059 or butyrolactone I (CDK2 inhibitor). In conclusion, estradiol-17 stimulates the proliferation of murine ES cells, and this action is mediated by MAPKs, CDKs, or protooncogenes. cyclin-dependent kinase; mitogen-activated protein kinase     

Peroxisome proliferator-activated receptor-gamma ligands regulate endothelial membrane superoxide production

Recently, we demonstrated that the peroxisome proliferator-activated receptor- (PPAR-) ligands, either 15-deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂) or ciglitazone, increased endothelial nitric oxide (·NO) release without altering endothelial nitric oxide synthase (eNOS) expression (4). However, the precise molecular mechanisms of PPAR--stimulated endothelial·NO release remain to be defined. Superoxide anion radical (O₂^–·) combines with ·NO to decrease·NO bioavailability. NADPH oxidase, which produces O₂^–·, and Cu/Zn-superoxide dismutase (Cu/Zn-SOD), which degrades O₂^–·, thereby contribute to regulation of endothelial cell·NO metabolism. Therefore, we examined the ability of PPAR- ligands to modulate endothelial O₂^–· metabolism through alterations in the expression and activity of NADPH oxidase or Cu/Zn-SOD. Treatment with 10 µM 15d-PGJ₂ or ciglitazone for 24 h decreased human umbilical vein endothelial cell (HUVEC) membrane NADPH-dependent O₂^–· production detected with electron spin resonance spectroscopy. Treatment with 15d-PGJ₂ or ciglitazone also reduced relative mRNA levels of the NADPH oxidase subunits, nox-1, gp91^phox (nox-2), and nox-4, as measured using real-time PCR analysis. Concordantly, Western blot analysis demonstrated that 15d-PGJ₂ or ciglitazone decreased nox-2 and nox-4 protein expression. PPAR- ligands also stimulated both activity and expression of Cu/Zn-SOD in HUVEC. These data suggest that in addition to any direct effects on endothelial·NO production, PPAR- ligands enhance endothelial·NO bioavailability, in part by altering endothelial O₂^–· metabolism through suppression of NADPH oxidase and induction of Cu/Zn-SOD. These findings further elucidate the molecular mechanisms by which PPAR- ligands directly alter vascular endothelial function. reduced nicotinamide adenine dinucleotide phosphate oxidase; copper/zinc superoxide dismutase; nitric oxide; endothelial cells     

The Role of Transmembrane Electrical Potential in Determining the Absorption Isotherm for Chloride in Potato

Measurements have been made of the electrical potential differencebetween the vacuoles of single potato tuber cells and externalCl^- solutions over the range 1–40 mM. With K⁺ as the counter-ion,the relationship between this transmembrane electrical potentialand external Cl^- concentration, for fresh cells at 20° C,was found to be one of decreasing negative polarity with increasingCl^- concentration (E at 1 mM Cl^- external = – 81 m V;change in E for a 10-fold change in external concentration,E₁₀ = 46 m V). The linearity of this relationship, apparenton a semi-logarithmic plot, was virtually unaltered by low temperature(0.5–2.5° C) or by previous ageing of the cells forperiods up to four days (indicating that metabolic ion absorptionis not an electrogenic process). When the counter-ion was maintainedat a constant high concentration (40 mM K⁺), the change in potentialover the Cl^- concentration range was only 4 m V, polarity becomingmore negative with increasing Cl^- concentration. With Ca⁺⁺ asthe counter-ion, the potential to external Cl^- concentrationrelationship was similar to that found in KCl solutions, exceptthat E₁₀ was only about 20 m V. Curves for the influx of C1^- to be expected on the basis ofthese electrochemical data alone have been shown to run closelyparallel to Cl^– absorption isotherms previously determinedexperimentally. This confirms the opinion, already formed onthe basis of theoretically derived values for passive Cl^- influx,that Cl^- uptake by both fresh and one-day-aged potato tissue,from KCl solutions and Cl^- solutions with a fixed high K⁺ concentration,is rate-determined at o° C by passive movement across theplasmalemma. Uptake of Cl^- by fresh tissue at 20° C appearsto be similarly regulated. No such parallelism was found between observed and expectedpatterns of Cl^- uptake from CaCl₂ solutions, or from KCl bytwo-day-aged tissue, and here factors in addition to the electrochemicalones must determine low temperature Cl^-uptake.     

Micropropagation of Pissardi Plum

An efficient medium for multiplication of Prunus cerasifera,J. F. Ehrh. cv. ‘Atropurpurea’, Pissardi plum, consistedof Linsmaier-Skoog basal medium (LS) supplemented with 3% sucrose,10mg 1^–1 N⁴-(²-isopentenyl) adenine (2iP), and 162 mg1^–1 phloroglucinol (Phl). Phl in the medium significantlyenhanced growth (P < 0.1 %) over cultures maintained on LSmedium with 10 mg 1^–1 2iP and no Phl. Plantlets were rootedon a half-strength Murashige-Skoog (MS) medium supplementedwith 0.2 mg 1^–1 indole-3-butyric acid (IBA). Prunus cerasifera, Pissardi plum, micropropagation, phloroglucinol, N⁶-(²-isopentenyl) adenine