Development of aldosterone-stimulation of short-circuit current across larval frog skin

Summary The short-circuit current (SCC) across isolated skin from bullfrog larvae in developmental stage XXI was small and insensitive to amiloride. Overnight incubation of this tissue with 10^-6 M aldosterone stimulated the SCC from 1.35±0.55 to 14.55±4.12 A·cm^-2 with 11.18±4.46 A·cm^-2 being blocked by 100 M amiloride. Histologic examination of aldosterone-treated skins revealed a separation of the apical cell layer from the underlying epidermis that was not seen in untreated preparations. The onset of amiloride-sensitive Na⁺ transport thus coincided with the exposure of the apical surface of newly differentiated epithelial cells. Similar results were obtained with skin from stage XXI larvae whose rate of metamorphosis had been stimulated by 10 g·l^-1 thyroxine (T₄) but not with skin from T₄-treated larvae in stages XIX and XX. Fluctuation analysis of the amiloride-sensitive SCC of the above preparations failed to show a consistent Lorentzian component in the power-density spectrum. Fluctuation analysis was possible on skins from larvae whose development had been accelerated by 7–9 days treatment with 10 g·l^-1 triiodothyronine (T₃). Aldosterone treatment of these tissues resulted in a significant increase in Na⁺ channel density.Abbreviations ASCC component of the short-circuit current (A·cm^-2) that is blocked by amiloride - fc frequency (Hz) at which the magnitude of the Lorenzian component of the power spectra is reduced by half - i current (pA) through individual amiloride-sensitive Na⁺ channels - I _Na+ amiloride-sensitive short-circuit current (A·cm^-2) that remains after treatment with a given amiloride concentration - k ₀₁ the rate constant (s^-1·M^-1) for the association of amiloride with Na⁺ channels - k ₁₀ rate constant (s^-1) for the dissociation of amiloride from Na⁺ channels - K _b magnitude of the power spectrum (A²·s·cm^-2) at a frequency of 1 Hz - KSCC short-circuit (A·cm^-2) current with K⁺ as the primary mucosal cation - M density of amiloride-sensitive Na⁺ channels in the apical cell membrane - SCC short-circuit current (A·cm^-2) - S _(f) magnitude of the power spectra (A²·s·cm^-2) at a given frequency - S ₀ the magnitude of the plateau region of the Lorentzian component of the power spectra (A²·s·cm^-2) - T ₃ Triiodothyronine - T ₄ Thyroxine     

Calcium influx at the plasmalemma of Chara corallina

Influx of ⁴⁵Ca into internodal cells of Chara corallina has been measured, using short uptake times, and a wash in ice-cold La³⁺-containing pondwater after the labelling period to overcome the difficulty of distinguishing extracellular tracer from that in the cell. Over 5–15 min the uptake was linear with time, through the origin. The basal influx from 0.1 mM Ca²⁺ externally was 0.25–0.5 pmol·cm^-2·s^-1, but some batches of cells showed higher fluxes. The influx was markedly stimulated by depolarisation in pondwater containing 20 mM K⁺. In cells in which the control flux was less than about 0.5 pmol·cm^-2·s^-1 there was no effect of 50 M nifedipine. In cells in which the control flux was greater than about 0.5 pmol·cm^-2·s^-1 (whether by natural variability, pretreatment, or by depolarisation in 20 mM K⁺), the flux was reduced by 50 M nifedipine to a value in the range 0.25–0.59 pmol·cm^-2·s^-1. It is suggested that two types of Ca-channel are probably involved, both opening on depolarisation, but only one sensitive to nifedipine. The flux was inhibited by 10 M BAY K 8644, which in animal cells more commonly opens Ca-channels. The apparent influx measured over long uptake times was much reduced, and the kinetics indicated filling a pool of apparent size about 1.45 nmol·cm^-2 with a halftime of about 38 min, probably representing cytoplasmic stores. It is argued that in spite of the very small pool of (free+bound) cytoplasmic Ca²⁺ the measured influx is a reasonable estimate of the influx at the plasmalemma.Abbreviations 0.4K-APW6 artificial pondwater, pH 6, containing 0.4 mM KCl - 20 K-APW6 artificial pondwater, pH 6, containing 20 mM KCl - Ca_o external Ca²⁺     

Transient state characteristics of adaptation to changes in light conditions for the cyanobacterium Oscillatoria agardhii

Transitions in growth irradiance level from 92 to 7 Em^-2 s^-1 and vice versa caused changes in the pigment contents and photosynthesis of Oscillatoria agardhii. The changes in chlorophyll a and C-phycocyanin contents during the transition from high to low irradiance (HL) were reflected in photosynthetic parameters. In the LH transition light utilization efficiencies of the cells changed faster than pigment contents. This appeared to be related to the lowering of light utilization efficiencies of photosynthesis. As a possible explanation it was hypothesized that excess photosynthate production led to feed back inhibition of photosynthesis. Time-scales of changes in the maximal rate of O₂ evolution were discussed as changes in the number of reaction centers of photosystem II in relation to photosynthetic electron transport. Parameters that were subject to change during irradiance transitions obeyed first order kinetics, but hysteresis occurred when comparing HL with LH transients. Interpretation of first order kinetic analysis was discussed in terms of adaptive response vs changes in growth rate.Non-standard abbreviations Chla chlorophyll a - CPC C-phycocyanin - PS II photosystem II - PS I photosystem I - RC II reaction center of photosystem II - P photosynthetic O₂-evolution - I irradiance, Em^-2 s^-1 - light utilization efficiency of cells, mmol O₂·mg dry wt^-1·h^-1/Em^-2 s^-1 - light utilization efficiency of photosynthetic apparatus, mol O₂·mol Chla ^-1·h^-1/Em^-2 s^-1 - P_max maximal rate of O₂ evolution by cells, mol O₂·mg dry wt^-1·h^-1 - P_max maximal rate of O₂ evolution by photosynthetic apparatus, mol O₂·mol·Chla ^-1·h^-1 - LL low light, E m^-2 s^-1 - HL high light, E m^-2 s^-1 - LH low to high light transition - HL high to low light transition - k specific rate of adaptation, h^-1 - specific growth rate, h^-1 - Q pool size of cell constituent, mol·mg dry wt^-1 - q net synthesis rate of cell constituent, mol·mg dry wt^-1·h^-1     

Orientation of reaction center complexes fromRhodobacter sphaeroides in proteoliposomes and the effect ofo-phenanthroline on electrogenesis during primary photochemical reaction

The orientation ofRhodobacter sphaeroides reaction center complexes (RC complexes) in proteoliposomal membranes was investigated by a direct electrometric method. Conditions were found that allow monitoring of only that RC complex fraction that is oriented with its donor side to the inner part of the proteoliposome. It is shown thato-phenanthroline, an inhibitor of electron transfer between primary (Q_A) and secondary (Q_B) quinone acceptors, can also inhibit the photoinduced Q_A reduction. The efficiency of this inhibition depends on the concentration of added ubiquinone. It is assumed that the laser flash-inducedo-phenanthroline inhibition of primary dipole (P-870⁺ · Q _A ^– ) formation is of a competitive nature.     

Solar heat gain in a desert rodent: unexpected increases with wind speed and implications for estimating the heat balance of free-living animals

We quantified metabolic power consumption as a function of wind speed in the presence and absence of simulated solar radiation in rock squirrels, Spermophilus variegatus, a diurnal rodent inhabiting arid regions of Mexico and the western United States. In the absence of solar radiation, metabolic rate increased 2.2-fold as wind speed increased from 0.25 to 4.0 m·s^-1. Whole-body thermal resistance declined 56% as wind speed increased over this range, indicating that body insulation in this species is much more sensitive to wind disruption than in other mammals. In the presence of 950 W·m^-2 simulated solar radiation, metabolic rate increased 2.3-fold as wind speed was elevated from 0.25 to 4.0 m·s^-1. Solar heat gain, calculated as the reduction in metabolic heat production associated with the addition of solar radiation, increased with wind speed from 1.26 mW·g^-1 at 0.25 m·s^-1 to 2.92 mW·g^-1 at 4.0 m·s^-1. This increase is opposite to theoretical expectations. Both the unexpected increase in solar heat gain at elevated wind speeds and the large-scale reduction of coat insulation suggests that assumptions often used in heat-transfer analyses of animals can produce important errors.Abbreviations absorptivity of coat to solar radiation - kinematic viscosity of air (mm²·s^-1) - reflectivity of coat to solar radiation - a r _B expected at zero wind speed (s·m^-1) - A _P projected surface area of animal on plane perpendicular to solar beam (cm²) - A _SKIN skin surface area (cm²) - b Coefficient describing change in r _B with change in square-root of wind speed (s^1.5·m^1.5) - d hair diameter (m) - d characteristic dimension of animal (m) - D _H thermal diffusivity of air (m²·s^-1) - E evaporative heat loss (W·m^-2) - I probability per unit coat depth that photon will strike hair - k constant equalling 1200 J·m^-3·°C^-1 - l _C coat depth m) - l _H hair length (m) - M metabolic rate (W·m^-2) - n density of hairs of skin (m^-2) - Q _A solar heat gain to animal (W·m^-2) - Q _I solar irradiance intercepted by animal (W·m^-2) - RQ respiratory quotient - r _A thermal resistance of boundary layer (s·m^-1) - r _B whole-body thermal resistance (s·m^-1) - r _E thermal resistance between animal surface and environment s·m^-1) - r _R radiative resistance (s·m^-1) - r _S sum of r _B and r _E at 0.25 m·s^-1 (s·m^-1) - r _T tissue thermal resistance s·m^-1) - T _AIR air temperature (°C) - T _B body temperature (°C) - T _E operative temperature of environment (°C) - T _ES standard operative temperature of environment (°C) - u wind speed (m·s^-1)     

Two sites of photoinhibition of the electron transfer in oxygen evolving and Tris-treated PS II membrane fragments from spinach

Photoinhibition was analyzed in O₂-evolving and in Tris-treated PS II membrane fragments by measuring flash-induced absorption changes at 830 nm reflecting the transient P680⁺ formation and oxygen evolution. Irradiation by visible light affects the PS II electron transfer at two different sites: a) photoinhibition of site I eliminates the capability to perform a stable charge separation between P680⁺ and Q_A ^- within the reaction center (RC) and b) photoinhibition of site II blocks the electron transfer from Y_Z to P680⁺. The quantum yield of site I photoinhibition (2–3×10^-7 inhibited RC/quantum) is independent of the functional integrity of the water oxidizing system. In contrast, the quantum yield of photoinhibition at site II depends strongly on the oxygen evolution capacity. In O₂-evolving samples, the quantum yield of site II photoinhibition is about 10^-7 inhibited RC/quantum. After selective elimination of the O₂-evolving capacity by Tris-treatment, the quantum yield of photoinhibition at site II depends on the light intensity. At low intensity (<3 W/m²), the quantum yield is 10^-4 inhibited RC/quantum (about 1000 times higher than in oxygen evolving samples). Based on these results it is inferred that the dominating deleterious effect of photoinhibition cannot be ascribed to an unique target site or a single mechanism because it depends on different experimental conditions (e.g., light intensity) and the functional status of the PS II complex.Abbreviations A₈₃₀ absorption change at 830 nm - P680 primary electron donor of PS II - PS II photosystem II - Mes 2(N-morpholino)ethansulfonic acid - Q_A, Q_B primary and secondary acceptors of PS II - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbohydrazide - FWHM fullwidth at half maximum - Ph-p-BQ phenyl-p-benzoquinone - PFR photon fluence rate - Pheo pheophytin - RC reaction center     

Organization and function of cytochromeb and ubiquinone in the cristae membrane of beef heart mitochondria

The arrangement and function of the redox centers of the mammalianbc ₁ complex is described on the basis of structural data derived from amino acid sequence studies and secondary structure predictions and on the basis of functional studies (i.e., EPR data, inhibitor studies, and kinetic experiments). Two ubiquinone reaction centers do exist—a QH₂ oxidation center situated at the outer, cytosolic surface of the cristae membrane (Q₀ center), and a Q reduction center (Q _i center) situated more to the inner surface of the cristae membrane. The Q₀ center is formed by theb-566 domain of cytochromeb, the FeS protein, and maybe an additional small subunit, whereas the Q _i center is formed by theb-562 domain of cytochromeb and presumably the 13.4kDa protein (QP-C). The Q binding proteins are proposed to be protein subunits of the Q reaction centers of various multiprotein complexes. The path of electron flow branches at the Q₀ center, half of the electrons flowing via the high-potential cytochrome chain to oxygen and half of the electrons cycling back into the Q pool via the cytochromeb path connecting the two Q reaction centers. During oxidation of QH₂, 2H⁺ are released to the cytosolic space and during reduction of Q, 2H⁺ are taken up from the matrix side, resulting in a net transport across the membrane of 2H⁺ per e^– flown from QH₂ to cytochromec, the H⁺ being transported across the membrane as H (H⁺ + e^–) by the mobile carrier Q. The authors correct their earlier view of cytochromeb functioning as a H⁺ pump, proposing that the redox-linkedpK changes of the acidic groups of cytochromeb are involved in the protonation/deprotonation processes taking place during the reduction and oxidation of Q. The reviewers stress that cytochromeb is in equilibrium with the Q pool via the Q _i center, but not via the Q₀ center. Their view of the mechanisms taking place at the reductase is a Q cycle linked to a Q-pool where cytochromeb is acting as an electron pump.     

Ion transport across leech integument

The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5±0.5 k· cm^-2) with an initial short-circuit current of 29.0±2.9 A·cm^-2. Removal of Na⁺ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50mol·l^-1) added to the basolateral solution, depressed the short-circuit current completely. The Na⁺ current saturated at a concentration of 90 mmol Na⁺·l^-1 in the apical solution (K _M=11.2±1.8 mmol·l^-1). Amiloride (100 mol·l^-1) on the apical side inhibited ca. 40% of the Na⁺ current and indicated the presence of Na⁺ channels. The dependence of Na⁺ current on the amiloride concentration followed Michaclis-Menten kinetics (K _i=2.9±0.4 mol·l^-1). The amiloride analogue benzamil had a higher affinity to the Na⁺ channel (K _i=0.7±0.2 mol·l^-1). Thus, Na⁺ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na⁺·l^-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8±2.3% of total short-circuit current. Higher Na⁺ concentrations lead to a decrease in amiloride-blockade short-circuit current. Sitmulation of the tissue with cyclic adenosine monophosphate (100 mol·l^-1) and isobutylmethylxanthine (1 mmol·l^-1) nearly doubled short-circuit current and increased amiloride-sensitive Na⁺ currents by 50%. By current fluctuation analysis we estimated single Na⁺ channel current (2.7±0.9 pA) and Na⁺ channel density (3.6±0.6 channels·m^-2) under control conditions. After cyclic adenosine monophosphate stimulation Na⁺ channel density increased to 5.4±1.1 channels·m^-2, whereas single Na⁺ channel current showed no significant change (1.9±0.2 pA). These data present a detailed investigation of an invertebrate epithelial Na⁺ channel, and show the similarities and differences to vertebrate Na⁺ channels. Whereas the channel properties are different from the classical vertebrate Na⁺ channel, the regulation by cyclic adenosine monophosphate seems similar. Stimulation of Na⁺ uptake by cyclic adenosine monophosphate is mediated by an increasing number of Na⁺ channels.Abbreviations slope of the background noise component - ADH antidiuretic hormone - cAMP cyclic adenosine monophosphate - f frequency - f _c coner frequency of the Lorentzian noise component - Hepes N-hydroxyethylpiperazine-N-ethanesulphonic acid - BMX isobutyl-methylxanthine - i _Na single Na⁺ channel current - I _Na max, maximal inhibitable Na⁺ current - I _SC short circuit current - K _i half maximal blocker concentration - K _M Michaelis constandard error of the mean - S _(f) power density of the Lorentzian noise component - S ₀ plateau value of the Lorentzian noise component - TMA tetramethylammonium - Trizma TRIS-hydroxymethyl-amino-methane - V _max maximal reaction velocity - V _T transepithelial potential - K half maximal blocker concentration     

Hydraulic resistance to radial water flow in growing hypocotyl of soybean measured by a new pressure-perfusion technique

Hydraulic resistances to water flow have been determined in the cortex of hypocotyls of growing seedlings of soybean (Glycine max L. Merr. cv. Wayne). Data at the cell level (hydraulic conductivity, Lp; half-time of water exchange, T _1/2; elastic modulus, ; diffusivity for the cell-to-cell pathway, D _c) were obtained by the pressure probe, diffusivities for the tissue (D _t) by sorption experiments and the hydraulic conductivity of the entire cortex (Lp_r) by a new pressure-perfusion technique. For cortical cells in the elongating and mature regions of the hypocotyls T _1/2=0.4–15.1 s, Lp=0.2·10^-5–10.0·10^-5 cm s^-1 bar^-1 and D _c=0.1·10^-6–5.5·10^-6 cm² s^-1. Sorption kinetics yielded a tissue diffusivity D _t=0.2·10^-6–0.8·10^-6 cm² s^-1. The sorption kinetics include both cell-wall and cell-to-cell pathways for water transport. By comparing D _c and D _t, it was concluded that during swelling or shrinking of the tissue and during growth a substantial amount of water moves from cell to cell. The pressure-perfusion technique imposed hydrostatic gradients across the cortex either by manipulating the hydrostatic pressure in the xylem of hypocotyl segments or by forcing water from outside into the xylem. In segments with intact cuticle, the hydraulic conductance of the radial path (Lp_r) was a function of the rate of water flow and also of flow direction. In segments without cuticle, Lp_r was large (Lp_r=2·10^-5–20·10^-5 cm s^-1 bar^-1) and exceeded the corticla cell Lp. The results of the pressure-perfusion experiments are not compatible with a cell-to-cell transport and can only the explained by a preferred apoplasmic water movement. A tentative explanation for the differences found in the different types of experiments is that during hydrostatic perfusion the apoplasmic path dominates because of the high hydraulic conductivity of the cell wall or a preferred water movement by film flow in the intercellular space system. For shrinking and swelling experiments and during growth, the films are small and the cell-to-cell path dominates. This could lead to larger gradients in water potential in the tissue than expected from Lp_r. It is suggested that the reason for the preference of the cell-to-cell path during swelling and growth is that the solute contribution to the driving force in the apoplast is small, and tensions normally present in the wall prevent sufficiently thick water films from forming. The solute contribution is not very effective because the reflection coefficient of the cell-wall material should be very small for small solutes. The results demonstrate that in plant tissues the relative magnitude of cell-wall versus cell-to-cell transport could dependent on the physical nature of the driving forces (hydrostatic, osmotic) involved.Abbreviations and symbols D _c diffusivity of the cell-to-cell pathway - D _t diffusivity of the tissue - radial flow rate per cm² of segment surface - Lp hydraulic conductivity of plasma-membrane - Lp_r radial hydraulic conductance of the cortex - T _1/2 half-time of water exchange between cell and surroundings - volumetric elastic modulus     

Calculation of leaf photosynthetic parameters from light-response curves for ecophysiological applications

Measurement of the light response of photosynthetic CO₂ uptake is often used as an implement in ecophysiological studies. A method is described to calculate photosynthetic parameters, such as the maximum rate of whole electron transport and dissimilative respiration in the light, from the light response of CO₂ uptake. Examples of the light-response curves of flag leaves and ears of wheat (Triticum aestivum cv. ARKAS) are shown.Abbreviations and symbols A net photosynthesis rate - D ¹ rate of dissimilative respiration occurring in the light - f loss factor - I incident PPFD - I effective absorbed PPFD - J rate of whole electron transport - J _m maximum rate of whole electron transport - p ^c intercellular CO₂ partial pressure - PPFD photosynthetic photon flux density - q effectivity factor for the use of light (electrons/quanta) - absorption coefficient - I ^* CO₂ compensation point in the absence of dissimilative respiration (bar) - II conversion factor for calculation of CO₂ uptake from the rate of whole electron transport - convexity factor Gas-exchange rates relate to the projective area and are given in mol·m^-2·s^-1. Electron-transport rates are given in mol electrons·m^-2·s^-1; PPFD is given in mol quanta·m^-2·s^-1.     

Ethanol production with Zymomonas mobilis with synthetic medium in batch operation

Ethanol was produced with Zymomonas mobilis Z6 (ATCC 29191), in batch culture with synthetic medium on glucose as substrate and in the presence of aspartate. The concentrations of glucose, phosphate, ammonium, ethanol and dissolved O₂ and CO₂ in the medium and O₂ and CO₂ in the outlet gas as well as the cell mass by culture fluorescence were measured on-line. Cell mass, glucose and aspartate concentrations were measured off-line. In the presence of a sufficient amount of aspartate, the ethanol inhibition effect can be reduced considerably. However, the improvement with yeast extract is more incisive. The relationship between the intensity of culture fluorescence and cell mass concentration is linear, if sufficient aspartate is present.List of Symbols ASP kg/m³ aspartate concentration - CTR kg/(m³ · h) CO₂ transfer rate - N, NH₄ kg/m³ nitrogen concentration from NH ₄ ⁺ - P kg/m³ product (ethanol) concentration - p% product (ethanol) yield - PO₄ kg/m³ phosphate concentration - Q _E kg/(kg · h) specific ethanol production rate - kg/(kg · h) specific nitrogen uptake rate from NH ₄ ⁺ - Q _P kg/(kg · h) specific phosphate uptake rate - Q _s kg/(kg · h) specific substrate (glucose) uptake rate - S kg/m³ glucose concentration - S _O kg/m³ initial glucose concentration - Y _x/s kg/kg yield coefficient - h^–1 specific growth rate     

Strain selection,medium development and scale-up of toyocamycin production by streptomyces chrestomyceticus

The batch productivity (Q _TM) of the production of the nucleoside antibiotic toyocamycin (TM) by Streptomyces chrestomyceticus was increased ten-fold by selection of a UV generated mutant, optimization of pH, increasing incubation temperature from 28 °C to 36 °C, and addition of soy oil. Initial high oxygen transfer rates stimulated Q _TM maxima two-fold. Antibiotic production by the mutant strain, U190, however, appeared more shear sensitive than the parent culture FCRF 341 with maximum antibiotic titer being inversely related to impellor tip velocity, T _v . For this reason, scale-up could not be done at constant P/V or constant volumetric oxygen transfer. Instead, programming of impeller speed was evaluated in order to maintain optimal impeller tip velocity during scale-up. It was found that a low constant T _v maintained in scale-up in geometrically similar vessels was most beneficial for duplication of optimal antibiotic productivity, Q _TM. Pilot fermentations (120 dm³ scale) were used to determine coefficients of Q _TM variation from oxygen uptake rate (OUR) and total CO₂ evolution data for monitoring of Q _TM variation during scale-up to the 12,000 dm³ scale. This technique allowed for on-line prediction of antibiotic titer and Q _TM from fermentor exhaust gas data.List of Symbols A scale constant - B shape constant - C location of maximum constant - D m impeller diameter (m) - H m liquid height (m) - OTR MmolO₂·(dm³)^–1min^–1 oxygen transfer rate - OUR MmolO₂·(dm³)^–1min^–1 oxygen uptake rate - PCV cm³ packed cell volume - P/V watts/dm³ volumetric power consumption - Q 1 · min^–1 corrected to standard conditions of temperature, pressure aeration rate - Q _TM g/(cm³ · h) or kg/(m³ · h) antibiotic productivity - T m tank diameter - T _mix s mixing time - T _v cm · s^–1 impeller tip velocity - TM g/cm³ Toyocamycin concentration - TNP Tricyclic nucleoside phosphate     

Photoinactivation of functional photosystem II and D1-protein synthesis in vivo are independent of the modulation of the photosynthetic apparatus by growth irradiance

To investigate whether the in-vivo photoinhibition of photosystem II (PSII) function by excess light is an intrinsic property of PSII, the maximal photochemical efficiency of PSII (Fv/Fm) and the content of functional PSII (measured by repetitive flash yield of oxygen evolution) were determined in leaves of pea (Pisum sativum L.), grown in 50 (low light), 250 (medium light), and 650 (high light) mol photons·m^–2·s^–1. The modulation of PSII functionality in vivo was induced in 1.1% CO₂ by varying either (i) the duration (0–2 h) of light treatment (fixed at 1800 mol photons· m^–2·s^–1) or (ii) irradiance (0–3200 mol photons·m^–2·s^–1) at a fixed duration (1 h), after infiltration of leaves with water (control), lincomycin (an inhibitor of chloroplast-encoded protein synthesis), or a combination of lincomycin with nigericin (an uncoupler), through the cut petioles of leaves of 22-to 24-d-old plants. The reciprocity law of irradiance and duration of illumination for PSII function in vivo (Park et al. 1995, Planta 196: 401–411) holds in all differently light-grown peas, demonstrating that inactivation of functional PSII depends on photon exposure (mol photons·m^–2), not on the rate of photon absorption. In vivo, PSII acts as an intrinsic photon counter and at higher photon exposures is inactivated following absorption of about 3 × 10⁷ photons. There is a functional heterogeneity of PSII in vivo with 25% less-stable PSIIs that are inactivated at low photon exposure, compared to 75% more-stable PSIIs regardless of modulation of the photosynthetic apparatus. We suggest that the less-stable PSIIs represent monomers located in the nonappressed granal margins, while the more-stable PSIIs are dimers located in the appressed grana membrane cores. The capacity for D1-protein synthesis was the same in all the light-acclimated peas and saturated at low light, indicating that D1-protein repair is also an intrinsic property of PSII. This accounts for the low intensity required for recovery of photoinhibition in sun and shade plants which is independent of light-harvesting antennae size or PSII/PSI stoichiometries.Abbreviations D1-protein psbA gene product - D2 protein psbD gene product - Fo chlorophyll fluorescence corresponding to open PSII reaction centres - Fv, Fm variable and maximum fluorescence after dark incubation, respectively - PS photosystem - Q_B secondary quinone electron acceptor Financial support for this research by the Department of Employment, Education and Training/Australian Research Council International Research Fellowships Program (Korea) is gratefully acknowledged.     

Gas vesicle formation and buoyancy regulation in Pelodictyon phaeoclathratiforme (Green sulfur bacteria)

Gas vesicle formation and buoyancy regulation in Pelodictyon phaeoclathratiforme strain BU1 (Green sulfur bacteria) was investigated under various laboratory conditions. Cells formed gas vesicles exclusively at light intensities below 5 mol · m^-2 · s^-1 in the stationary phase. No effect of incubation temperature or nutrient limitation was observed. Gas space of gas vesicles occupied always less than 1.2% of the total cell volume. A maximum cell turgor pressure of 330 kPa was determined which is comparable to values determined for cyanobacterial species. Since a pressure of at least 485 kPa was required to collapse the weakest gas vesicles in Pelodictyon phaeoclathratiforme, short-term regulation of cell density by the turgor pressure mechanism can be excluded.Instead, regulation of the cell density is accomplished by the cease of gas vacuole production and accumulation of carbohydrate at high light intensity. The carbohydrate content of exponentially growing cells increased with light intensity, reaching a maximum of 35% of dry cell mass above 10 mol · m^-2 · s^-1. Density of the cells increased concomitantly. At maximum density, protein and carbohydrate together accounted for 62% of the total cell ballast. Cells harvested in the stationary phase had a significantly lower carbohydrate content (8–12% of the dry cell mass) and cell density (1010–1014 kg · m^-3 with gas vesicles collapsed) which in this case was independent of light intensity. Due to the presence of gas vesicles in these cultures, the density of cells reached a minimum value of 998.5 kg · m^-3 at 0.5 mol · m^-2 · s^-1.The cell volume during the stationary phase was three times higher than during exponential growth, leading to considerable changes in the buoyancy of Pelodictyon phaeoclathratiforme. Microscopic observations indicate that extracellular slime layers may contribute to these variations of cell volume.     

Changes in Photosystem II fluorescence in Chlamydomonas reinhardtii exposed to increasing levels of irradiance in relationship to the photosynthetic response to light

The effects of a 60 min exposure to photosynthetic photon flux densities ranging from 300 to 2200 mol m^–2s^–1 on the photosynthetic light response curve and on PS II heterogeneity as reflected in chlorophyll a fluorescence were investigated using the unicellular green alga Chlamydomonas reinhardtii. It was established that exposure to high light acts at three different regulatory or inhibitory levels; 1) regulation occurs from 300 to 780 mol m^–2s^–1 where total amount of PS II centers and the shape of the light response curve is not significantly changed, 2) a first photoinhibitory range above 780 up to 1600 mol m^–2s^–1 where a progressive inhibition of the quantum yield and the rate of bending (convexity) of the light response curve can be related to the loss of Q_B-reducing centers and 3) a second photoinhibitory range above 1600 mol m^–2s^–1 where the rate of light saturated photosynthesis also decreases and convexity reaches zero. This was related to a particularly large decrease in PS II centers and a large increase in spill-over in energy to PS I.Abbreviations Chl chlorophyll - DCMU 3,(3,4-dichlorophenyl)-1,1-dimethylurea - F_M maximal fluorescence yield - F_pl intermediate fluorescence yield plateau level - F₀ non-variable fluorescence yield - F_v total variable fluorescence yield (F_M-F₀) - initial slope to the light response curve, used as an estimate of initial quantum yield - convexity (rate of bending) of the light response curve of photosynthesis - LHC light-harvesting complex - P_max maximum rate of photosynthesis - PQ plastoquinone - Q photosynthetically active photon flux density (400–700 nm, mol m^–2s^–1) - PS photosystem - Q_A and Q_B primary and secondary quinone electron acceptor of PS II     

The effect of glucose on chloride uptake by Chlorella

Active Cl^- uptake by Chlorella fusca was examined by using ³⁶Cl as a label. Under light/air conditions chloride influx from a 2.4·10^-5 M solution was 4.0±0.04 nmol m^-2s^-1. After 70±10 min a stationary 380±40 fold accumulation was reached. In dark/air and dark/argon influx and accumulation were reduced to 25±6%, respectively, 5±1.5% of the light/air control. Cl^- uptake had a broad optimum around pH 7 and showed saturation kinetics with a K _M of 1.25·10^-5 M and a v _max of 7.0 nmol m^-2s^-1 in light/air. Br^- inhibited Cl^- uptake strongly, J^-, ClO ₄ ^- , SO ₄ ^2- , and NO ₃ ^- had no inhibitory effect. Inhibitor studies with carbonyl cyanide m-chlorophenylhydrazone and N,N-dicyclohexylcarbodiimide resulted in a good correlation between Cl^- uptake and ATP level. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea and darkness reduced transport activity without affecting the ATP level.The magnitudes of the pH gradient and the membrane potential across the cell membrane were determined and/or estimated under different conditions. It could be shown that in Chlorella Cl^- transport cannot proceed via secondary active H⁺/Cl^- cotransport. In addition, 2H⁺/Cl^- cotransport seems unlikely for energetic reasons. On the basis of the results of this and the following study, a primary active ATP-driven Cl^-/OH^- exchange pump is proposed.Abbreviations CCCP carbonyl cyanide m-chlorophenylhyd razone - DCCD N,N-dicyclohexylcarbodiimide - DCMU 3-(3.4-dichlorophenyl)-1.1-dimethylurea - DMO 5,5-dimethyloxazolidine-2,4-dione - Hepes N-2-hydroxyethylpiperazine-N ethane-sulfonic acid - POPOP 1.4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2.5-diphenyloxazole To whom correspondence should be addressed     

Acclimation of barley to changes in light intensity: photosynthetic electron transport activity and components

Barley seedlings (Hordeum vulgare L. Boone) were grown at 20°C with 16 h/8 h light/dark cycle of either high (H) intensity (500 mole m^-2 s^-1) or low (L) intensity (55 mole m^-2 s^-1) white light. Plants were transferred from high to low (H L) and low to high (L H) light intensity at various times from 4 to 8 d after leaf emergence from the soil. Primary leaves were harvested at the beginning of the photoperiod. Thylakoid membranes were isolated from 3 cm apical segments and assayed for photosynthetic electron transport, Photosystem II (PS II) atrazine-binding sites (Q_B), cytochrome f(Cytf) and the P-700 reaction center of Photosystem I (PS I). Whole chain, PS I and PS II electron transport activities were higher in H than in L controls. Q_B and Cytf were elevated in H plants compared with L plants. The acclimation of H L plants to low light occurred slowly over a period of 7 days and resulted in decreased whole chain and PS II electron transport with variable effects on PS I activity. The decrease in electron transport of H L plants was associated with a decrease in both Q_B and Cytf. In L H plants, acclimation to high light occurred slowly over a period of 7 days with increased whole chain, PS I and PS II activities. The increase in L H electron transport was associated with increased levels of Q_B and Cytf. In contrast to the light intensity effects on Q_B levels, the P-700 content was similar in both control and transferred plants. Therefore, PS II/PS I ratios were dependent on light environment.Abbreviations Asc ascorbate - BQ 2,5-dimethyl-p-benzoquinone - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCIP 2,6-dichlorophenolindophenol - H control plants grown under high light intensity - H L plants transferred from high to low light intensity - L low control plants grown under low light intensity - L H plants transferred from low to high light intensity - MV methyl viologen - P-700 photoreaction center of Photosystem I - Q_B atrazine binding site - TMPD N,N,N,N-tetramethyl-p-phenylenediamine Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC. Paper No. 11990 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643, USA.     

Photoinhibition and repair in Dunaliella salina acclimated to different growth irradiances

The light-dependent rate of photosystem-II (PSII) damage and repair was measured in photoautotrophic cultures of Dunaliella salina Teod. grown at different irradiances in the range 50–3000 mol photons · m^–2· s^–1. Rates of cell growth increased in the range of 50–800 mol photons·m^–2·s^–1, remained constant at a maximum in the range of 800–1,500 mol photons·m^–2 ·s^–1, and declined due to photoinhibition in the range of 1500–3000 mol photons·m^–2·s^–1. Western blot analyses, upon addition of lincomycin to the cultures, revealed first-order kinetics for the loss of the PSII reaction-center protein (D1) from the 32-kDa position, occurring as a result of photodamage. The rate constant of this 32-kDa protein loss was a linear function of cell growth irradiance. In the presence of lincomycin, loss of the other PSII reaction-center protein (D2) from the 34-kDa position was also observed, occurring with kinetics similar to those of the 32-kDa form of D1. Increasing rates of photodamage as a function of irradiance were accompanied by an increase in the steady-state level of a higher-molecular-weight protein complex ( 160-kDa) that cross-reacted with D1 antibodies. The steady-state level of the 160-kDa complex in thylakoids was also a linear function of cell growth irradiance. These observations suggest that photodamage to D1 converts stoichiometric amounts of D1 and D2 (i.e., the D1/D2 heterodimer) into a 160-kDa complex. This complex may help to stabilize the reaction-center proteins until degradation and replacement of D1 can occur. The results indicated an intrinsic half-time of about 60 min for the repair of individual PSII units, supporting the idea that degradation of D1 after photodamage is the rate-limiting step in the PSII repair process.Abbreviations Chl chlorophyll - PSI photosystem I - PSII photosystem II - D1 the 32-kDa reaction-center protein of PSII, encoded by the chloroplast psbA gene - D2 the 34-kDa reactioncenter protein of PSII, encoded by the chloroplast psbD gene - Q_A primary electron-accepting plastoquinone of PSII The work was supported by grant 94-37100-7529 from the US Department of Agriculture, National Research Initiative Competitive Grants Program.     

Analysis of oxygen evolution during photosynthetic induction and in multiple-turnover flashes in sunflower leaves

Exchange of CO₂ and O₂ and chlorophyll fluorescence were measured in the presence of 360 1 · 1^–1 CO₂ in nitrogen in Helianthus annuss L. leaves which had been preconditioned in the dark or at a photon flux density (PFD) of 24 mol · m^–2 · s^–1 either in 21 or 0% O₂. An initial light-dependent O₂ outburst of 6 mol · m^–2 was measured after aerobic dark incubation. It was attributed to the reduction of electron carriers, predominantly plastoquinone. The maximum initial rate of O₂ evolution at PFD 8000 mol · m^–2 · s^–1 was 170 mol · m^–2 · s^–2 or about four times the steady CO₂-and light-saturated rate of photosynthesis. Fluorescence measurements showed that the rate was still acceptor-limited. Fast O₂ evolution ceased after electron carriers were reduced in the dark-adapted leaf, but continued for a short time at the lower rate of 62 mol · m^–2 · s^–1 in the light-adapted leaf. The data are interpreted to show that enzymes involved in 3-phosphoglycerate reduction are dark-inhibited, but were fully active in low light. In a dark-adapted leaf, respiratory CO₂ evolution continued under nitrogen; it was partially inhibited by illumination. Prolonged exposure of a leaf to anaerobic conditions caused reducing equivalents to accumulate. This was shown by a slowly increasing chlorophyll fluorescence yield which indicated the reduction of the PSII acceptor Q_A in the dark. When the leaf was illuminated, no O₂ evolution was detected from short light pulses, although transient O₂ production was appreciable during longer light pulses. This indicates that an electron donor (pool size about 2–3 e/PSII reaction center) became reduced in the dark and the first photons were used to oxidise this donor instead of water.Abbreviations Chl chlorophyll - CRC carbon reduction cycle - GAPDH NADP-glyceraldehyde-phosphate dehydrogenase - PFD photon flux density - PGA 3-phosphoglycerate - RuBP ribulose bisphosphate - TCA tricarboxylic acid cycle To whom correspondence should be addressedThis work received support by the Estonian Academy of Sciences, the Gottfried-Wilhelm-Leibniz Program of the Deutsche For-schungsgemeinschaft and the Sonderforschungsbereich 251 of the University of Würzburg.     

Characterization of esophageal desalination in the seawater eel,Anguilla japonica

To characterize mechanisms of esophageal desalination, osmotic water permeability and ion fluxes were measured in the isolated esophagus of the seawater eel. The osmotic permeability coefficient in the seawater eel esophagus was 2·10^-4 cm·s^-1. This value was much lower than those in tight epithelial, although the eel esophagus is a leaky epithelium with a tissue resistance of 77 ohm·cm^-2. When the esophagus was bathed in normal Ringer solutions on both sides no net ion and water fluxes were observed. However, when mucosal NaCl concentration was increased by a factor of 3, Na⁺ und Cl^- ions were transferred from mucosa to serosa (desalination). If only Na⁺ or Cl^- concentration in the mucosal fluid was increased by a factor of 3, net Na⁺ and Cl^- fluxes were reduced to 30–40%, indicating that 60–70% of the net Na⁺ and Cl^- fluxes are coupled mutually. The coupled NaCl transport seems to be effective in desalting the luminal high NaCl. The remaining 30–40% of the total Na⁺ and Cl^- fluxes seems to be due to a simple diffusion, because these components are independent of each other and follow their electrochemical gradients, and also because these fluxes remain even after treatment with NaCN or ouabain. A half of the coupled NaCl transport could be explained by a Na⁺/H⁺–Cl^-/HCO ₃ ^- double exchanger on the apical membrane of the esophageal epithelium, because mucosal amiloride and 4.4-diisothiocyanatostilbene-2,2-disulphonic acid inhibited the net Na⁺ and Cl^- fluxes by approximately 30%. The other half of the coupled NaCl transport, which follows their electrochemical gradients, still remains to be explained.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NMDG N-methyl-d-glucosamine - P _Cl Cl^- permeability coefficient - PD transepithelial potential difference - P _Na Na⁺ permeability coefficient - P _osm osinotic permeability coefficient - TALH thick ascending limb of Henle's loop