Ozone increases the permeability of isolated pea chloroplasts

The effect of short-term exposure of chloroplasts isolated from the leaves of Pisum sativum to high concentrations of ozone was examined. The inhibitory effect of O₃ on endogenous photophosphorylation was apparently related to an increased permeability of the chloroplast limiting membranes induced by ozone exposure. A 5 min treatment with 50 ppm O₃ reduced the reflection coefficient of meso-erythritol from 0.84 to 0.58 and that of glycerol from 0.26 to 0.03. Such decreases in reflection coefficients indicate that ozone caused a marked increase in the permeability of the limiting membranes of the chloroplasts, which may result from an oxidation of membrane lipids. The decrease in the reflection coefficient of meso-erythritol was proportional both to ozone concentration (up to 30 ppm for 5 min of bubbling) and to time (up to 5 min at 30 ppm). Extrapolating these results to lower concentrations and longer times, ozone injury should be possible for a 2 hr exposure of plants to 0.3 ppm ozone, as is indeed the case.     

Membrane Permeability Modeling: Kedem–Katchalsky vs a Two-Parameter Formalism

The analysis of experiments for the purpose of determining cell membrane permeability parameters is often done using the Kedem–Katchalsky (KK) formalism (1958). In this formalism, three parameters, the hydraulic conductivity (L_p), the solute permeability (P_s), and a reflection coefficient (ς), are used to characterize the membrane. Sigma was introduced to characterize flux interactions when water and solute (cryoprotectant) cross the membrane through a common channel. However, the recent discovery and characterization of water channels (aquaporins) in biological membranes reveals that aquaporins are highly selective for water and do not typically cotransport cryoprotectants. In this circumstance, sigma is a superfluous parameter, as pointed out by Kedem and Katchalsky. When sigma is unneeded, a two-parameter model (2P) utilizing onlyL_pandP_sis sufficient, simpler to implement, and less prone to spurious results. In this paper we demonstrate that the 2P and KK formalism yield essentially the same result (L_pandP_s) when cotransporting channels are absent. This demonstration is accomplished using simulation techniques to compare the transport response of a model cell using a KK or 2P formalism. Sigma is often misunderstood, even when its use is appropriate. It is discussed extensively here and several simulations are used to illustrate and clarify its meaning. We also discuss the phenomenological nature of transport parameters in many experiments, especially when both bilayer and channel transport are present.     

On simultaneous transport of water and solute through plant cell membranes: Evidence for the absence of solvent drag effect and insensitivity of the reflection coefficient

The simultaneous efflux of tritiated water and ¹⁴C labelled ethanol from inner epidermal cells of the bulb scale of Allium cepa was measured with a specially designed efflux chamber. It was found that water and ethanol moved essentially independently. Rates of efflux of tritiated water and ¹⁴C ethanol were essentially the same in the presence or absence of a simultaneous influx of water. Using the same technique the efflux of tritiated water from the epidermal cells was measured during a simultaneous flow of nonlabelled ethanol. When tritiated water and ethanol moved in opposite directions, the water permeability values became slightly reduced depending upon the concentration of ethanol. When ethanol and tritiated water moved in the same direction, however, no effect on water permeability values could be detected. These results are best explained by the molecular theory of diffusion across lipid bilayer membranes, and are consistent with the above findings of lack of interaction between water and ethanol as they are transported across the cell membrane. In another study, the solute permeability coefficients (K_s) for non-electrolytes such as urea and methyl urea were measured by plasmolyzing the epidermal cells and transferring them to equimolal solutions of urea and methyl urea. This method was also used to measure the reflection coefficient (σ) for these nonelectrolytes. The K_s values for methyl urea were 16 times greater than the ones for urea. The values of σ for both of these solutes, however, were very close to 1. Using the K_s data available in the literature for the subepidermal cells of the Pisum sativum stem basis, the σ values were calculated for malonamide, glycerol, methyl urea, ethyl urea, dimethyl urea, and formamide. Again the K_s values for these nonelectrolytes varied by several orders of magnitude, whereas all σ values were found to be close to 1. These findings point out that σ is an insensitive parameter and that K_s, the solute permeability constant, has to be used for characterizing solute transport through the membrane. The present study shows that fast (e.g. ethanol, formamide) as well as slowly permeating molecules do not interact with water as they are transported across the cell membrane. Aqueous pores for the simultaneous transport of water and solutes, therefore, are absent in the plant cell membranes investigated here.     

Permeability characteristics of the basement membrane (basal lamella) of the intestine of Ascaris suum

Permeability characteristics have been determined for isolated ribbons of the basement membrane of the intestine ofAscaris suum. The solute permeability coefficient (P_c) was measured for a series of hydrophobic, nonionic molecules of graded molecular size. The geometric pore area per unit path length (A_o/Δx) was estimated to be 24.0 cm from the diffusion rates for the various solute molecules. A filtration coefficient (L_p) of 18.1×10^?12 cm⁵/dyne-sec was determined by a method that employs osmotic pressure. The preceding values were used to calculate an average pore radius of 24.0 A for the membrane. The unstirred layer was estimated to be 30μm thick from measurements of the change in the rate of diffusion of water across the membrane with change in the rate of perfusion. The preceding values were used to calculate a reflection coefficien (σ), effective permeability coefficient (ω′), and a permeability coefficient (ω). The results support the view that this basement membrane functions as a filter and selective barrier to diffusion of constituents of the worm's body fluid.     

Oxidative gating of water channels (aquaporins) in Chara by hydroxyl radicals

Hydroxyl radicals (*OH) as produced in the Fenton reaction (Fe²⁺ + H₂O₂ = Fe³⁺ + OH^– + *OH) have been used to reversibly inhibit aquaporins in the plasma membrane of internodes of Chara corallina. Compared to conventional agents such as HgCl₂, *OH proved to be more effective in blocking water channels and was less toxic to the cell. When internodes were treated for 30 min, cell hydraulic conductivity (Lp) decreased by 90% or even more. This effect was reversed within a few minutes after removing the radicals from the medium. In contrast to HgCl₂, radical treatment reduced membrane permeability of small lipophilic organic solutes (ethanol, acetone, 1‐propanol, and 2‐propanol) by only 24 to 52%, indicating some continued limited movement of these solutes across aquaporins. The biggest effect of *OH treatment on solute permeability was found for isotopic water (HDO), which largely used water channels to cross the membrane. Inhibition of aquaporins reduced the diffusional water permeability (P_d) by about 70%. For the organic test solutes, which mainly use the bilayer to cross the membrane, channel closure caused anomalous (negative) osmosis; that is, cells had negative reflection coefficients (σ_s) and were transiently swelling in a hypertonic medium. From the ratio of bulk (Lp or osmotic permeability coefficient, P_f) to diffusional (P_d) permeability of water, the number (N) of water molecules that align in water channels was estimated to be N = P_f/P_d = 46 (on average). Radical treatment decreased N from 46 to 11, a value still larger than unity, which would be expected for a membrane lacking pores. The gating of aquaporins by *OH radicals is discussed in terms of a direct action of the radicals when passing the pores or by an indirect action via the bilayer. The rapid recovery of inhibited channels may indicate an easy access of cytoplasmic antioxidants to closed water channels. As hydrogen peroxide is a major signalling substance during different biotic and abiotic stresses, the reversible closure of water channels by *OH (as produced from H₂O₂ in the apoplast in the presence of transition metals such as Fe²⁺ or Cu⁺) may be downstream of the H₂O₂ signalling. This may provide appropriate adjustments in water relations (hydraulic conductivity), and a common response to different kinds of stresses.     

Water permeability of Betula periderm

The water permeability of periderm membranes stripped from mature trees of Betula pendula Roth was investigated. The diffusion of water was studied using the system water/membrane/water, and transpiration was measured using the system water/membrane/water vapor. Betula periderm consists of successive periderm layers each made up of about 5 heavily suberized cell layers and a varying number of cell layers that are little suberized, if at all. It is shown that these layers act as resistances in series. The permeability coefficient of the diffusion of water (P _d) can be predicted with 79% accuracy from the reciprocal of the membrane weight (x in mg cm^-2) by means of the linear equation P _d=14.69·10^-7 x-0.73·10^-7. For example, the P _d of a periderm membrane having a weight of 10 mg cm^-2 (approx. 250 m thick) is 7.4·10^-8 cm s^-1, which is comparable to the permeability of cuticles. This comparison shows that on a basis of unit thickness, Betula periderm is quite permeable to water as cuticles have the same resistance with a thickness of only 0.5 to 3 m. It is argued that this comparatively high water permeability of birch periderm is due to the fact that middle lamellae and the primary walls of periderm cells are not at all, or only incompletely suberized and, therefore, form a hydrophilic network within which the water can flow. This conclusion is based on the following observations: (1) Middle lamellae and primary walls stain strongly with toluidine blue, which shows them to be polar. (2) If silver ions are added as tracer for the flow of water, they are found only in the middle lamellae, primary walls, and in plasmodesmata, while no silver can be detected in the suberized walls. (3) Permeability coefficients of transpiration strongly depend on water activity. This shows conclusively that water flows across Betula periderm via a polar pathway. It is further argued that liquid continuity is likely to be maintained under all physiological conditions in the network formed by middle lamellae and primary walls. On the other hand, the lumina of periderm cells, intercellular air spaces in the lenticels, and even the pores in the suberized walls (remainders of plasmodesmata) will drain at a humidity of 95% and below. Due to the presence of intercellulars the permeability coefficient of lenticels is much greater than that of the periderm. A substantial amount of the total water, therefore, flows as vapor through lenticels even though they cover only 3% of the surface.Abbreviations PM perideron membrane - P _d permeability coefficient for diffusion of water - P _tt permeability coefficient of transpiration - MES (N-morpholino)ethane sulfonic acid     

A model for radial water transport across plant roots

Summary A model based on the canal theory (Katou andFurumoto 1986 a, b) is proposed for the absorption of solute and water at the root periphery. The present canal model in the periphery and the model which was previously proposed for the exudation in the stele (Katou et al. 1987), are organized into a model for radial transport across excised plant roots, in the light of anatomical and physiological knowledge of maize roots. The canal equations for both canals are numerically solved to give quite a good explanation for the observed exudation of maize roots. It is found that the regulation of solute transport has a primary importance in the regulation of water transport across excised roots. The internal cell pressure of the symplast adjusts the water absorption at the root periphery to the water secretion into the vessels. There seems no need for this explanation of the radial water transport across roots to assume cell membranes with low reflection coefficient or variable water permeability. It would seem that the apoplast wall layers play a crucial role in metabolic control of water transport in roots as well as in hypocotyls.Abbreviations J _s ^ex* the theoretically estimated rate of solute exudation per unit surface area of model maize roots - J that of volume exudation per unit surface area of model maize roots - the reflection coefficient of the cell membrane against solutes     

Effects of Ozone Fumigation on Photosynthesis and Membrane Permeability in Leaves of Spring Barley, Meadow Fescue, and Winter Rape

Seedlings of spring barley, meadow fescue, and winter rape were fumigated with 180 μg kg⁻¹ of ozone for 12 d, and effect of O₃ on photosynthesis and cell membrane permeability of fumigated plants was determined. Electrolyte leakage and chlorophyll fluorescence were measured after 6, 9, and 12 d of fumigation, while net photosynthetic rate (P _N) and stomatal conductance (g _s) were measured 9 d after the start of ozone exposure. O₃ treatment did not change membrane permeability in fescue and barley leaves, while in rape a significant decrease in ion leakage was noted within the whole experiment. O₃ did not change the photochemical efficiency of photosystem 2 (PS2), i.e., F_v/F_m, and the initial fluorescence (F₀). The values of half-rise time (t_1/2) from F₀ to maximal fluorescence (F_m) decreased in fescue and barley after 6 and 9 d of fumigation. P _N decreased significantly in ozonated plants, in the three species. The greatest decrease in P _N was observed in ozonated barley plants (17 % of the control). The ozone-induced decrease in P _N was due to the closure of stomata. Rape was more resistant to ozone than fescue or barley. Apparently, the rape plants show a large adaptation to ozone and prevent loss of membrane integrity leading to ion leakage. This revised version was published online in August 2006 with corrections to the Cover Date.     

Assessing the microbial oxidative stress mechanism of ozone treatment through the responses of Escherichia coli mutants

Aims:  To investigate the effect of the oxidative stress of ozone on the microbial inactivation, cell membrane integrity and permeability and morphology changes of Escherichia coli. Methods and Results:  Escherichia coli BW 25113 and its isogenic mutants in soxR, soxS, oxyR, rpoS and dnaK genes were treated with ozone at a concentration of 6 μg ml^?1 for a period up to 240 s. A significant effect of ozone exposure on microbial inactivation was observed. After ozonation, minor effects on the cell membrane integrity and permeability were observed, while scanning electron microscopy analysis showed slightly altered cell surface structure. Conclusions:  The results of this study suggest that cell lysis was not the major mechanism of microbial inactivation. The deletion of oxidative stress–related genes resulted in increased susceptibility of E. coli cells to ozone treatment, implying that they play an important role for protection against the radicals produced by ozone. However, DnaK that has previously been shown to protect against oxidative stress did not protect against ozone treatment in this study. Furthermore, RpoS was important for the survival against ozone. Significance and Impact of the Study:  This study provides important information about the role of oxidative stress in the responses of E. coli during ozonation.     

Investigations into biophysical regulation mechanisms of physiological water exchange of the Nitella translucens cells with the surroundings

In the present paper, we have presented a theoretical discussion of specific biophysical mechanisms pertaining to the regulation (by means of internal factors) of physiological water exchange with the water medium by the cells of the Nitella translucens. The investigation has demonstrated that these cells have a capacity for water exchange regulation through changes in the values of transport parameters of the cell membrane (i.e. the filtration coefficient L_p and the reflection coefficient σ), as well as changes in the concentration C_si (inside the cells) of the osmotically active solute.     

The role of the lateral intercellular spaces and solute polarization effects in the passive flow of water across the rabbit gallbladder

Summary Osmotic water flows were measured acrossin vitro preparations of the rabbit gallbladder by a gravimetric technique. The bladders exhibited asymmetrical osmotic behavior, in which theL _p (hydraulic conductivity) for water flow from mucosa to serosa was up to four times greater than theL _p for water flow in the opposite direction. This result is similar to the effects of osmotic gradients on ion and nonelectrolyte permeability reported in the first paper. As in the case of solute permeability, these changes inL _p are accounted for by changes in the dimensions of the lateral intercellular spaces of the epithelium. These spaces are thus a final common pathway for the movement of both solutes and water across the epithelium. We also observed osmotic flow transients in which the initialL _p was about an order of magnitude greater than the steady stateL _p . These transients are largely explained by solute polarization in the unstirred layers adjacent to the epithelial membranes. A comparison between streaming potentials and water flows showed that streaming potentials are directly proportional to the rate of flow only over a limited range. These observations are readily explained on the basis of structural changes and solute polarization effects. Finally, the routes of water flow across epithelia are discussed in the light of our observations.     

Effect of dDAVP on basolateral cell surface water permeability in the outer medullary collecting duct

We report a novel approach for assessing the volume of living cells which allows quantitative, high-resolution characterization of dynamic changes in cell volume while retaining the cell functionality. The aim of this study was to evaluate the short-term effect of vasopressin on basolateral cell surface water permeability in the outer medullary collecting duct (OMCD). The permeability of the basolateral cell membrane was determined in the tubules where the apical membrane was blocked with oil injected into the lumen. The apparent coefficient of water permeability (P _f) was evaluated by measuring the cell swelling after the step from hypertonic to isotonic medium (600 mosm to 300 mosm). Desmopressin (dDAVP) induced an increase of the basolateral P _f from 113.7±8.5 μm/s in control cells to 186.6±11.4 μm/s in micro-dissected fragments of the OMCD incubated in vitro (10⁻⁷ M dDAVP, 30 min at 37 °C) (P<0.05). Mercury caused pronounced inhibition of basolateral water permeability (26.0±6.9 μm/s; P<0.05). The effect of mercury (1.0 mM HgCl₂) was reversible: after washing the fragments with PBS for 20 min, P _f values were restored to the control levels (125.0±9.5 μm/s). The results of the study indicate the existence of a mechanism controlling the osmotic water permeability of the basolateral cell membrane in the OMCD epithelium.     

Development of a microfluidic device for determination of cell osmotic behavior and membrane transport properties

An understanding of cell osmotic behavior and membrane transport properties is indispensable for cryobiology research and development of cell-type-specific, optimal cryopreservation conditions. A microfluidic perfusion system is developed here to measure the kinetic changes of cell volume under various extracellular conditions, in order to determine cell osmotic behavior and membrane transport properties. The system is fabricated using soft lithography and is comprised of microfluidic channels and a perfusion chamber for trapping cells. During experiments, rat basophilic leukemia (RBL-1 line) cells were injected into the inlet of the device, allowed to flow downstream, and were trapped within a perfusion chamber. The fluid continues to flow to the outlet due to suction produced by a Hamilton Syringe. Two sets of experiments have been performed: the cells were perfused by (1) hypertonic solutions with different concentrations of non-permeating solutes and (2) solutions containing a permeating cryoprotective agent (CPA), dimethylsulfoxide (Me₂SO), plus non-permeating solute (sodium chloride (NaCl)), respectively. From experiment (1), cell osmotically inactive volume (V_b) and the permeability coefficient of water (L_p) for RBL cells are determined to be 41% [n = 18, correlation coefficient (r²) of 0.903] of original/isotonic volume, and 0.32 ± 0.05 μm/min/atm (n = 8, r² > 0.963), respectively, for room temperature (22 °C). From experiment (2), the permeability coefficient of water (L_p) and of Me₂SO (P_s) for RBL cells are 0.38 ± 0.09 μm/min/atm and (0.49 ± 0.13) × 10⁻³ cm/min (n = 5, r² > 0.86), respectively. We conclude that this device enables us to: (1) readily monitor the changes of extracellular conditions by perfusing single or a group of cells with prepared media; (2) confine cells (or a cell) within a monolayer chamber, which prevents imaging ambiguity, such as cells overlapping or moving out of the focus plane; (3) study individual cell osmotic response and determine cell membrane transport properties; and (4) reduce labor requirements for its disposability and ensure low manufacturing costs.     

Exposure of Dunaliella tertiolecta to Lead and Aluminum: Toxicity and Effects on Ultrastructure

The growth response of the marine alga Dunaliella tertiolecta to different concentrations of lead and aluminum was investigated. Both metals had a stimulatory effect at low concentration and an inhibitory effect at high concentration (hormesis). The IC₂₅ values of lead are 8.43, 7.29, and 6.74 mg L⁻¹ for 24, 48, and 72 h, respectively. The corresponding values for aluminum are 30.54, 22.42, and 18.16 mg L⁻¹. Although it seems that the two metals are not directly toxic to the alga at the concentrations found in the environment, as implied by the IC₂₅ values and the environmental concentrations of the metals, low concentrations of both metals, alone and in combination, affected the ultrastructure. The growth of batch-grown cells exposed to 0.5 mg L⁻¹ lead and aluminum, alone and combined, during the 24-h exponential phase was investigated. The same cells were also examined under an electron microscope to determine the biological effects of the two metals on the ultrastructure. The most obvious effects of lead were disrupted thylakoidal membranes, accumulated polyphosphate bodies and vacuoles, and lead precipitates on the cell surface. These ultrastructural alterations were partially present in aluminum-treated and lead–aluminum-treated cells. In joint exposure, the most important change was the lysis of the cell membrane. Aluminum and lead seem to act synergistically on the cell membrane leading to cell membrane lysis.     

Enhancing hypericin production of Hypericum perforatum cell suspension culture by ozone exposure

Accumulation of secondary metabolites is one of the common reactions of plants to ozone exposure in nature. To investigate the effect of ozone on the production of desired compounds of plant cell cultures, we assayed hypericin production of Hypericum perforatum suspension cell cultures treated with different doses of ozone at different culture phases. The results show that hypericin contents of the cells treated with 60 to 180 nL L^?1 ozone are significantly higher than those of the control, showing that ozone exposure may stimulate hypericin synthesis. Hypericin production of the cells treated with ozone at exponential phase is higher than that of lag and stationary phase, which suggests that exponential phase cell cultures are more responsive to ozone exposure than lag and stationary phase cells. The highest hypericin production is obtained by the cells exposed to 90 nL L^?1 ozone at late exponential phase for 3 h, being about fourfold of the control. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Water and ion permeability of a native collagen membrane

With the help of a ribonucleoprotein it is possible to precipitate collagen in a layer of fibers with a 700 Å period. As collagen is a constituent of many membrane systems in the body, it seemed interesting to investigate the permeability of ions and water through a native collagen membrane.The experiments were carried out with the help of an acryl glass apparatus, where an osmotic pressure, a hydrostatic pressure difference or both can be maintained between the two bulk phases separated by the membrane. The diffusion coefficients for NaCl and KCl were found to be comparable with those in other biological membranes (D_s = 9 · 10⁻⁷cm² · s⁻¹) whereas there is difference of more than three orders of magnitude in the hydraulic permeability (L_p = 6 cm⁴ · J⁻¹ · s⁻¹).Volume flow measurements caused by an osmotic gradient indicated that the reflection coefficient for NaCl and KCl is very small. In hydrostatic pressure experiments, the membrane shows a preferred direction for volume flows which seems to have something to do with the mode of preparation of the membrane.     

The diffusional and osmotic water permeability of the hindgut cuticle in larval Sarcophaga bullata

The diffusional water permeability (P_D) and the pressure filtration coefficient (L_P) of isolated larval hindgut cuticle of the fleshfly, Sarcophaga bullata, were measured using tracer techniques coupled with a simple mathematical model system based on equations of non-equilibrium thermodynamics. Data obtained from the model system were matched to experimental tracer data by means of a mathematical optimization scheme. The following parameter values were obtained: P_D for tritiated water = 1.02 × 10^?6 cm-sec^?1, and L_P = 9.18 × 10^?11 cm³-dyn^?1-sec^?1. These results are now being used to determine reflection coefficients (σ's) and solute mobilities (ω's) for the cuticle system in an attempt to gain an understanding of the mechanisms controlling solute and water movements across the hindgut wall.     

Ozone-mediated cytotoxicity after short-term exposure and its relation to the production of cellular metabolites (NO,H2O2)

Alveolar macrophages secrete numerous mediators, playing an important role in host defence. Among these mediators, nitric oxide (NO) and hydrogen peroxide (H₂O₂) are both involved in bactericidal killing and trigger the release of other cellular metabolites. We have analyzed the effect of an atmosphere polluted with ozone (0.03–0.5 ppm v/v) on the monocytic cell line THP-1, as a model for alveolar macrophages,in vitro. NO and H₂O₂ were chosen to evaluate cell response to ozone. Cell injury was evaluated using lactate dehydrogenase (LDH) liberation into the medium. An exposure to 0.5 ppm ozone proved to be more toxic to the cells, than 0.1 or 0.03 ppm, evidenced by more LDH being liberated and cytotoxicity reaching values up to 64%. For all ozone concentrations, H₂O₂ production reached a peak value after 10–15 min of exposure, after which the concentration of extracellular H₂O₂ production diminished rapidly. The highest NO concentrations were measured with 0.5 ppm ozone, reaching a maximum value of 1460 nmol/L per 5×10⁶ cells, which is 1.55 times higher than for nonexposed cells. Lower concentrations barely induced higher NO concentrations compared to nonexposed cells. The results indicate that ozone effects not only the viability of human monocytes but also the release of antibacterial and defense signaling molecules and suggest that ozone-mediated cytotoxicity may be related to the secretion of NO and H₂O₂. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ozone-induced damage of Escherichia coli K-12

Escherichia coli K-12 transformed with pACYC184 plasmid DNA was exposed to ozone (O₃) in aqueous solution. The damage to the membrane, protein, plasmid DNA, and cell survival were investigated. Cell viability was unaffected by short-term O₃ exposure (1–5 min) but membrane permeability was compromised as indicated by protein and nucleic acid leakage and lipid oxidation. The intracellular components, protein and DNA, remained intact. With longer durations of O₃ exposure (up to 30 min) cell viability decreased with a more significant increase in lipid oxidation and protein and nucleic acid leakage. The proteins leaking out were further oxidized by O₃. The total intracellular proteins run on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and plasmid DNA run on agarose gel, showed progressive degradation corresponding to the decrease in cell viability. The data indicate that membrane components are the primary targets of O₃ damage with subsequent reactions involving the intracellular components, protein and DNA. Received: 18 Apirl 1996 / Received revision: 26 July 1996 / Accepted: 5 August 1996