Changes In Cell Permeability Research Articles

Supernatants from dengue virus type-2 infected macrophages induce permeability changes in endothelial cell monolayers.

The ability of dengue virus-infected human monocyte-derived macrophages to induce permeability changes in primary human umbilical vein endothelial cells was investigated. Supernatants from dengue virus type 2-infected monocyte-derived macrophages increased permeability in human umbilical vein endothelial cell monolayers without inducing endothelial cell infection. Production of permeabilising activity from monocyte-derived macrophages occurred after the peak of progeny virus release. TNF-alpha, a known inducer of endothelial cell permeability, was released from dengue virus infected monocyte-derived macrophages but its release did not coincide with release of endothelial cell permeabilising activity. Permeability induction was enhanced by pre-incubation with supernatants from infected monocyte-derived macrophages harvested at the time of peak release of TNF-alpha and infectious virus. Thus, supernatants from dengue virus-infected monocyte-derived macrophages contain factors that increase human umbilical vein endothelial cell permeability, but this is not accompanied by endothelial cell infection or directly correlated with release of dengue virus or TNF-alpha from monocyte-derived macrophages. This model system can be used for further in vitro analysis of mechanisms that may relate to capillary leakage and the development of dengue haemorrhagic fever/dengue shock syndrome.

Induction of permeability changes and death of vertebrate cells is modulated by the virulence of Entamoeba spp. isolates

Although Entamoeba histolytica is capable of inducing an apoptotic response in vertebrate cells in vitro (Cell. Microbiol. 2 (2000) 617), it is not known whether vertebrate cell death requires direct amoeba–vertebrate cell contact or simply the presence of amoebae in the area of the vertebrate cells. In addition, Entamoeba spp. vary in their virulence and pathogenicity. The potential effects of these critical parameters also have not been elucidated. We tested the virulent HM-1:IMSS isolate and the non-virulent Rahman isolate of E. histolytica, and the non-virulent E. dispar CYNO16:TPC isolate against two vertebrate cell lines, HeLa and Chinese hamster ovary cells in vitro using ethidium homodimer as a fluorescent indicator of changes in vertebrate cell permeability. Fluorescence appeared in vertebrate cell nuclei within approximately 2–3 min of contact between HM-1 amoebae and vertebrate cells independent of vertebrate cell type. However, vertebrate cells in the immediate vicinity of but not contacted by HM-1 amoebae were not affected. In contrast, although both E. histolytica Rahman and E. dispar CYNO16 amoebae moved freely among and contacted vertebrate cells, the nuclei of the vertebrate cells never fluoresced implying that the cells remained alive and impermeant to the ethidium homodimer. This is the first demonstration that direct contact between virulent amoebae and vertebrate cells is required to kill vertebrate cells and that the process is restricted to virulent Entamoeba isolates. An understanding at the molecular level of the processes involved could help to reduce the pathology associated with this parasite.

Changes in distribution spaces and cell permeability caused by ATP in the rat liver.

Cellular and extracellular volume changes caused by ATP were investigated in the liver as well as the possible formation of diffusion barriers, which could be responsible for some of its metabolic effects. The experimental system was the bivascularly perfused rat liver. [(14)C]Sucrose and [(3)H]water were simultaneously injected into either the portal vein or the hepatic artery. Mean transit times, distribution spaces, variances and linear superimpositions were calculated. In the portal system, ATP reduced the transit time in the great vessels, had little or no effect on sinusoidal and cellular spaces, but impaired the flow-limited distribution of both [(14)C]sucrose and [(3)H]water. In the arterial bed ATP infused into either the portal vein or the hepatic artery produced vasodilation and increased the aqueous extra-sucrose space. These effects were inhibited by Nomega-nitro-L-arginine methyl ester infused into the hepatic artery. Sucrose and extra-sucrose space changes caused in the arterial bed by portally infused ATP are most probably analogous to the transhepatic vasodilation effect already described for the rabbit liver. Impairment of flow-limited distribution of tracers in the sinusoidal bed indicates that ATP induces the formation of permeability barriers, which could be responsible for some of its metabolic effects.

Clostridium perfringens type A enterotoxin forms mepacrine-sensitive pores in pure phospholipid bilayers in the absence of putative receptor proteins

Clostridium perfringens enterotoxin (CPE) is an important cause of food poisoning with no significant homology to other enterotoxins and its mechanism of action remains uncertain. Although CPE has recently been shown to complex with tight junction proteins, we have previously demonstrated that CPE increases ionic permeability in single Caco-2 cells using the whole-cell patch-clamp technique, thereby excluding any paracellular permeability. In this paper we demonstrate that CPE forms pores in synthetic phospholipid membranes in the absence of receptor proteins. The properties of the pores are consistent with CPE-induced permeability changes in Caco-2 cells suggesting that CPE has innate pore-forming ability.

Cytolytic peptides induce biphasic permeability changes in mammalian cell membranes

The cytolytic peptides melittin and gramicidin S are naturally occurring agents that provide a comparative model for studies of complement, immunotoxin and cell-mediated membrane permeability. Most attempts to characterize cytolytic peptides have used model membrane systems including phospholipid vesicles or erythrocytes. Membrane vesicles permit the use of self-quenching concentrations of fluorescent permeability markers, while erythrocytes release measurable hemoglobin. Attempts at measuring early membrane permeability changes in nucleated mammalian cells have been limited. To measure the kinetics of mammalian cell membrane permeability changes induced by cytolytic peptides, we developed a 96-well fluorescence cytolysis assay using the cytoplasmic fluorescent dye calcein as the membrane permeability marker. To facilitate rapid assessment of membrane permeability, trypan blue was added to the assay solution to quench (a) released fluorescence and (b) retained intracellular fluorescence. Trypan blue also provided a complementary visual assessment of cell viability. Using this assay, a detailed kinetic analysis demonstrated permeability of the cell membranes within seconds of exposure to the cytolytic peptides. The rapid permeabilization of the cell membranes was confirmed by flow cytometry using the calcium indicator dye fluo-3. The assay also demonstrated a second slower phase of marker release over the next several hours. The fluorescence cytolysis assay was able to reliably detect the biphasic permeability changes associated with the melittin and gramicidin S peptides suggesting the potential utility of this assay in the assessment of other cytolytic agents.

The potential of active metabolites of antihistamines in the management of allergic disease.

Since the discovery of histamine in the ®rst decade of the 20th century, there has been increasing evidence that this biogenic amine, synthesized predominantly in mast cells and basophils, is released in in ammatory processes and plays a key role in the pathogenesis of urticaria and allergic airway diseases, including asthma, rhinitis, and urticaria (1±3). Studies have demonstrated that histamine levels are signi®cantly increased in the bronchoalveolar lavage (BAL) of symptomatic asthmatics, compared with asymptomatic asthmatics (4, 5), and that allergen challenge leads to a marked increase in the levels of histamine in asthmatics (6, 7). Similarly, studies have demonstrated that allergen challenge also leads to signi®cant increases in the levels of histamine in the nasal secretions of patients with allergic rhinitis (8±10). A comparison of patients with chronic urticaria and healthy subjects has also demonstrated that the former release greater amounts of histamine, both spontaneously and after antigen provocation (11±13). In addition, some studies have demonstrated that the responsiveness of the skin to histamine is slightly increased in patients with urticaria (11). Mechanistic studies have demonstrated that the action of histamine arises from its interaction with one of at least three speci®c histamine receptors, H1, H2, and H3, located on various histamine-responsive target tissues. Interaction of histamine with the H1 receptor leads primarily to smooth-muscle contraction, vasodilatation and hypotension, increased vascular permeability with formation of tissue oedema, increased respiratory mucus secretion, and parasympathetic nerve stimulation (14, 15). These effects can subsequently lead to bronchial obstruction in asthma; nasal blockage, sneezing, itching, and discharge in rhinitis; and itchy skin wheals/ ares in urticaria. At the cellular level, histamine may also act on in ammatory cells and lead to release of mediators, such as leukotrienes and cytokines, and increased expression of class II antigens (HLA-DR) and ICAM-1 on epithelial cells (16, 17), effects which could, in turn, exacerbate the pathology and symptoms of allergic disease. In contrast, effects attributable to H2 receptors include augmentation of gastric acid secretion and increases in gastric and respiratory mucus secretion (14). H3 receptors have been implicated in autocrine regulation of histamine synthesis and release from nerve tissue (14).

Patients' expressions of complaints as a predictor of the course of acute hepatitis A

Objective: Physical complaints, emotional states, liver, and immune parameters were investigated as possible indicators of the course of hepatitis A. Methods: Forty-seven patients with hepatitis A were studied by means of the Giessen-Complaint-Inventory (GBB) and the Berlin Mood Questionnaire (BSF), as well as by taking into account their typical liver parameters and the following immune parameters: αInterferon (αIFN), soluble Interleukin 2 receptor (sIL-2R), Interleukin 6 (IL-6), Interleukin 1 receptor antagonist (IL-1Ra), and Immunoglobulin M or G (IgM, IgG). Two hundred twenty-nine medical students and a representative German sample (n = 1557, Braehler et al.) served as controls. Results: We found that the initial degree of change in permeability of the liver cells, complaints about abdominal symptoms, and extent of depressive mood were able to predict the length of hospital stay. Patients with the initial, more pronounced liver damage, as well as patients who articulated less subjective impairment proved to have a longer course of illness. Those patients who needed a significantly longer time for recovery report, at admission, very few complaints—fewer than even the normal population. We were not, however, able to demonstrate a significant difference in the investigated immune parameters. Conclusion: A certain denial tendency seems to be harmful with respect to the recovery process and the immunological competence in the course of hepatitis A.

Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor.

1. Extracellular ATP acting on P2X7 receptors opens a channel permeable to small cations, creates an access pathway for the entry of larger molecular weight dyes, and causes cell death. We used whole-cell recording and fluorescence microscopy to measure the time courses of ionic currents, uptake of the propidium dye YO-PRO-1, and membrane disruption, in human embryonic kidney (HEK293) cells expressing the rat P2X7 receptor. 2. The ATP analogue 2', 3'-O-(benzoyl-4-benzoyl)-ATP (30 microM) induced membrane blebbing within 30-40 s of sustained application; this was 5-10 times slower when extracellular sodium was replaced by larger cations. 3. Fluorescence of YO-PRO-1 was detectable within 3 s, and the uptake reached a steady rate within 10-20 s; YO-PRO-1 uptake was greatly enhanced by removing extracellular sodium. 4. Electrophysiological measurements of current reversal potentials with intracellular sodium and extracellular cations of different sizes showed that the ionic channel progressively t'2+LE0i%-i"dilated during 10-20 s to a diameter greater than 1 nm (10 A). With short agonist applications (3-5 s) the pore dilatation and YO-PRO-1 uptake were reversible and repeatable. 5. Polyethylene glycols having molecular weights >= 5000 blocked the increase in cation permeability, YO-PRO-1 uptake and membrane blebbing. 6. We conclude that maximum P2X7 receptor activation causes an exponential dilatation of the ion channel with a time constant of 25 s to a final diameter of 3-5 nm from an initial minimum pore diameter of 0.8 nm.

Reactive oxygen species activate a Ca2+-dependent cell death pathway in the unicellular organism Trypanosoma brucei brucei

Here we examine a cell death process induced by reactive oxygen species (ROS) in the haemoflagellate Trypanosoma brucei brucei. Ca2+ distribution in cellular compartments was measured with stable transformants expressing aequorin targeted to the cytosol, nucleus or mitochondrion. Within 1.5 h of ROS production, mitochondrial Ca2+ transport was impaired and the Ca2+ barrier between the nuclear envelope and cytosol was disrupted. Consequently the mitochondrion did not accumulate Ca2+ efficiently in response to an extracellular stimulus, and excess Ca2+ accumulated in the nucleus. The terminal transferase deoxytidyl uridine end labelling assay revealed that, 5 h after treatment with ROS, extensive fragmentation of nuclear DNA occurred in over 90% of the cells. Permeability changes in the plasma membrane did not occur until an additional 2 h had elapsed. The intracellular Ca2+ buffer, EGTA acetoxymethyl ester, prevented DNA fragmentation and prolonged the onset of changes in cell permeability. Despite some similarities to apoptosis, nuclease activation was not a consequence of caspase 3, caspase 1, calpain, serine protease, cysteine protease or proteasome activity. Moreover, trypanosomes expressing mouse Bcl-2 were not protected from ROS even though protection from mitochondrial dysfunction and ROS have been reported for mammalian cells. Overall, these results demonstrate that Ca2+ pathways can induce pathology in trypanosomes, although the specific proteins involved might be distinct from those in metazoans.

Reactive oxygen species activate a Ca2+-dependent cell death pathway in the unicellular organism Trypanosoma brucei brucei

Here we examine a cell death process induced by reactive oxygen species (ROS) in the haemoflagellate Trypanosoma brucei brucei. Ca2+ distribution in cellular compartments was measured with stable transformants expressing aequorin targeted to the cytosol, nucleus or mitochondrion. Within 1.5 h of ROS production, mitochondrial Ca2+ transport was impaired and the Ca2+ barrier between the nuclear envelope and cytosol was disrupted. Consequently the mitochondrion did not accumulate Ca2+ efficiently in response to an extracellular stimulus, and excess Ca2+ accumulated in the nucleus. The terminal transferase deoxytidyl uridine end labelling assay revealed that, 5 h after treatment with ROS, extensive fragmentation of nuclear DNA occurred in over 90% of the cells. Permeability changes in the plasma membrane did not occur until an additional 2 h had elapsed. The intracellular Ca2+ buffer, EGTA acetoxymethyl ester, prevented DNA fragmentation and prolonged the onset of changes in cell permeability. Despite some similarities to apoptosis, nuclease activation was not a consequence of caspase 3, caspase 1, calpain, serine protease, cysteine protease or proteasome activity. Moreover, trypanosomes expressing mouse Bcl-2 were not protected from ROS even though protection from mitochondrial dysfunction and ROS have been reported for mammalian cells. Overall, these results demonstrate that Ca2+ pathways can induce pathology in trypanosomes, although the specific proteins involved might be distinct from those in metazoans.

Sublethal Damage during Cryopreservation of Rainbow Trout Sperm

Although cellular damage during cryopreservation of freshwater fish spermatozoa has been reported in several studies, there is a lack of correlation between this damage and the fertility rates of eggs using postthawed milt. The apparent lack of such correlation may be due to other undetected sublethal cryodamage, which could affect the cell functionality and viability. This may be extremely important for freshwater fish spermatozoa whose ability to fertilize the egg requires dilution in water or hypoosmotic solutions, an hazardous environment for the cells. This study tested the change in cell permeability during cryopreservation, using Hoechst 33258 to assess cell permeability. The permeability of spermatozoa at different times after dilution in several hypoosmotic media were investigated. In the first trial, fresh semen, sperm diluted in freezing media (CPT), and freeze/thawed semen were studied. Three CPT were tested (Me2SO, DMA, and methanol). In the second trial, the addition of egg yolk as a membrane stabilizer was investigated. Samples were frozen at −20°C/min in a programmable cooler and thawed in a 25°C water bath. Dilution in the CPTs slightly increased the susceptibility of cells to damage but freezing/thawing caused a dramatic increase in the fragility of cells, which were killed in a few seconds after their contact with the hypoosmotic solutions. Egg yolk provided a significant protection to the membrane, allowing the cells a greater and more prolonged survival in the fertilization media. Samples frozen with Me2SO displayed the best results. These results are consistent with the achieved fertility rates that demonstrated sublethal cryodamage in the function of the sperm membrane that was not detected by standard procedures.

HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region.

To analyze the mechanisms of release and the extracellular fate of the HIV-1 Tat protein and to determine the Tat domain binding to the extracellular matrix. Release of Tat was studied by pulse-chase experiments with Tat-transfected COS-1 cells in the presence or absence of different serum concentrations, temperatures and drugs inhibiting the classical secretion pathway or endo-exocytosis, such as brefeldin A and methylamine. The binding of extracellular Tat to heparan sulfate proteoglycans (HSPG) was determined by using trypsin, heparin or heparinase in pulse-chase experiments, by gel shift and competition assays with radiolabeled heparin, and by heparin-affinity chromatography. The mapping of the Tat binding site to heparin was defined by functional assays of rescue of Tat-defective HIV-1 proviruses. Tat is released in the absence of cell death or permeability changes. Tat release is dependent upon the temperature and serum concentration, and it is not blocked by brefeldin A or methylamine. After release, a portion of the protein remains in a soluble form whereas the other binds to extracellular matrix (ECM)-associated HSPG. The HSPG-bound Tat can be retrieved into a soluble form by heparin, heparinase or trypsin. Binding to heparin is competed out by heparin-binding factors such as basic fibroblast growth factor (bFGF), and it is mediated by the Tat basic region which forms a specific complex with heparin which blocks HIV-1 rescue by exogenous Tat and allows purification of a highly biologically active protein. These results demonstrate that Tat exits from intact cells through a leaderless secretion pathway which shares several features with that of acid FGF or bFGF. The released Tat binds to HSPG through its basic region and this determines its storage into the ECM, as occurs for bFGF.

Metabolic and permeability changes caused by thiamine deficiency in immortalized rat brain microvessel endothelial cells

The possible involvement of blood–brain barrier (BBB) breakdown in the pathogenesis of thiamine deficiency encephalopathy was investigated in RBE4 cells, an immortalized rat brain endothelial cell line. The effects of thiamine deficiency produced by addition of pyrithiamine and by reduction of thiamine in the culture medium, on the metabolism and permeability of the RBE4 monolayer was examined. Pyrithiamine treatment in low thiamine medium (M199) for 7 days caused cytotoxic effects on RBE4 cells at all concentrations (10–50 μg/ml). Pyrithiamine caused a concentration- and time-dependent decrease in MTT reduction and a significant increase in glucose consumption and lactate production compared to controls. Pyrithiamine treatment for 3 days caused a significant decrease in MTT reduction at 50 μg/ml only. In contrast, increased glucose consumption and lactate production by the RBE4 cells was observed after treatment for 3 days with concentrations of 25 μg/ml pyrithiamine and above. The permeability of RBE4 cell monolayers to [ 14C]sucrose ( M w 342), but not FITC-dextran ( M w 4000) was significantly increased by treatment with pyrithiamine concentrations of 25 μg/ml and above for 3 days. These effects were not accompanied by detectable changes in F-actin distribution or content, although F-actin content was significantly reduced by 7 days exposure to pyrithiamine. These results suggest that metabolic and permeability changes in thiamine-deficient RBE4 cells may be important early events in thiamine-deficiency encephalopathy. The relative role of the BBB in the pathogenesis of thiamine deficiency is discussed.

HCO3−efflux and the regulation of the intracellular Cipool size inSynechococcusUTEX 625

Cyanobacterial cells of Synechococcus UTEX 625 were suspended in increasing extracellular inorganic carbon (Ci, up to 1 mM) under steady state photosynthesis conditions in a cuvette connected to a mass spectrometer. Photosynthetic CO2fixation, net CO2, and net HCO3−transport increased with increased extracellular Ciup to 100 μM [C1], with HCO3−transport contributing the most to the maintenance of the internal Cipool. When the extracellular [Ci] was increased above 100 μM, CO2, fixation and net CO2transport increased toward their maximum rates but net HCO3−transport progressively declined. At an extracellular [Ci] of 1 mM, the net HCO3−transport rate was only 15% of its value at 100 μM extracellular [Ci]. Under steady state photosynthesis conditions, the gross HCO3−transport rate remained at its maximum value at extracellular [Ci] above 100 μM either when CO2transport was precluded (i.e., in the presence of 200 μM Na2S) or when HCO3−transport was measured with H14CO3−and silicone oil filtration. As HCO3−efflux, calculated using the permeability coefficient determined at low [Ci], could not account fully for the decrease of the net HCO3−transport rate, Ciefflux and CO2, efflux were measured at high extracellular [Ci] during the initial period of darkness. With increasing intracellular [Ci] Ciefflux over intracellular [Ci] increased linearly but CO2, efflux over intracellular [Ci] was constant, hence the HCO3−efflux obtained by the difference between the Ciand the CO2efflux increased hyperbolically. Using HCO3−and CO2, efflux, we calculated that the permeability coefficient of the cell envelope for HCO3−but not for CO2increased with increasing intracellular [Ci]. When the HCO3−efflux was measured under steady state Ciflux conditions in the light, the permeability coefficient of the cell envelope for HCO3−also increased with increasing intracellular [Ci]. Reasons for the change in whole cell permeability to HCO3−at high extracellular [Ci] are discussed. Key words: Synechococcus UTEX 625, blue green algae, cyanobacteria, active HCO3−transport, HCO3−leakage, Ciconcentrating mechanism.

水耕における溶存Ｏ２濃度制御下のキュウリ植物の吸水

For analyzing the effect of dissolved O2 concentration on root water uptake in hydroponics, the stem base water flux in cucumber plants (Cucumis sativus L.) was measured by heat flux control method (HFC) in “a” newly developed system for controlling dissolved O2 concentration. At an air temperature of 25°C, a relative humidity of 70%, a PPFD of 300μmol m-2 s-1 (a photoperiod of 12 h) and a root temperature of 25°C, dissolved O2 concentration was controlled at set values of 0.01, 0.10 and 0.20 mM with an accuracy of ±0.005 mM. At each dissolved O2 concentration, the water uptake rate drastically increased by lighting the plant, and water uptake reduced in proportion to decrease in dissolved O2 concentration: Water uptakes per day at 0.20, 0.10 and 0.01 mM were 235, 186 and 164 g, respectively. This fact suggests that root water uptake in hydroponics is depressed at lower dissolved O2 concentrations by change in membrane permeability of root cells through respiration-dependent processes.

Endotoxin-induced changes of endothelial cell viability and permeability: Protective effect of a 21-aminosteroid

Endotoxic shock results in endothelial cell dysfunction and oedema formation. Endotoxin decreased in a concentration-dependent fashion endothelial cell viability with maximum effects by adding 10 μg/ml lipopolysaccharide for 48 h (47 ± 15% and 22 ± 2.6% of control cells in the presence or absence of foetal calf serum, respectively). Furthermore, incubation (10 h) with lower concentrations of lipopolysaccharide (1 μg/ml) significantly increased endothelial cell permeability to 250% compared to control values. The 21-aminosteroid U-74389G (10 μM) prevented the cytotoxic effect of lipopolysaccharide as well as the lipopolysaccharide-induced increase in endothelial cell permeability. By contrast, the glucocorticoid methylprednisolone was less effective even at higher concentrations (100 μM). The effect of lipopolysaccharide is possibly due to oxidative stress and/or membrane destabilization rather than to the induction of inflammatory mediators, because of the reduced efficacy of the glucocorticoid.

Changes in growth, osmotic potential and cell permeability of wheat cultivars under salt stress

10-d-old wheat seedlings were grown hydroponically in presence and absence of 100 mM NaCl for 7 d. Salt stress decreased growth of shoots and roots of both cultivars; fresh mass of sensitive cultivar being more affected. NaCl increased membrane permeability to urea, methylurea and ethylurea and decreased membrane partiality in root cortex cells of sensitive cultivar. Neither parameter changed by NaCl in resistant cultivar. NaCl treatment decreased water permeability and osmotic potential in both cultivars; sensitive cultivar was more affected. The results extends our previous data that cell membrane properties are different in salt sensitive and resistant genotypes and so cell permeability could be a potential trait indicating salt tolerance.

The role of intracellular free calcium in motor neuron disease

The intracellular calcium (Ca 2+) concentrations of motoneurons can be altered by the influx of Ca 2+ into the cell by the opening of voltage-dependent Ca 2+ channels and ligand-gated channels linked to Ca 2+ influx, especially by the N- methyl- d-aspartate (NMDA) type of excitatory amino acid receptor. Intracellular Ca 2+ concentration is also affected by the release of Ca 2+ buffered in mitochondria and endoplasmic reticulum. Evidence that motoneurons may be selectively vulnerable to Ca 2+-induced cell death include the following observations: (i) the presence of excitatory amino acid receptors on the cell membranes of motoneurons, some of which would permit Ca 2+ influx (e.g. NMDA receptors); (ii) the availability of the presynaptic terminal for antibody-mediated effects leading to changes in cell permeability and Ca 2+ influx; and (iii) the limited amounts of intracellular Ca 2+-binding proteins such as calbindin D 28K and parvalbumin in motoneurons. Elevation of intracellular free Ca 2+ may also be a common event in a number of independent mechanisms leading to motoneuron death in motor neuron disease.

Membrane Lipid Metabolism, Cell Permeability, and Ultrastructural Changes in Lightly Processed Carrots

ABSTRACTWe monitored changes in phospholipid (PL), steryl lipid, and glycolipid classes, cell permeability, and ultrastructure in wound‐stressed tissues (shreds and disks) of carrots (Daucus carota L. ‘Apache’), stored UD to 10 days at 10°C and 95% RH. Total PL rose 47% ten days after shredding, with phosphatidylcholine decreasing and phosphatidic acid increasing in relative abundance. Acylated sterol glycoside doubled after 2 days. Leakage of UV‐absorbing metabolites from disks increased by 45% between 1 and 3 days storage. Extensive, storage‐dependent accumulation of endoplasmic reticulum and attached ribosomes within vascular parenchyma cells occurred 10 days after wounding. Thus net synthesis of membrane lipid components occurred together with increases in permeability and the accumulation of phosphatidic acid. Membrane degradation and repair processes likely coexist during prolonged storage of lightly processed carrots.

Effect of miconazole on the structure and function of plasma membrane of Candida albicans

The primary mode of action of azoles is the inhibition of cytochrome P-450 dependent 14α-demethylase, a key enzyme in ergosterol biosynthesis in fungi. Our results demonstrated that Candida albicans cells grown in the presence of 10 μg ml−1 of miconazole (miconazole-grown), do not possess ergosterol in their plasma membranes and this ergosterol depletion leads to a drastic change in membrane fluidity as shown by fluorescence polarization measurements and unsaturation index. There was an increase in membrane order in miconazole-grown cells and a reduced rate of uptake of amino acids. We also checked for membrane permeability changes in normal mid-log phase cells (normal-grown) in short incubations (10 min) with 10 μg ml−1 miconazole (miconazole-incubated). Interestingly, the amino acid uptake rates except that Gly were not affected significantly in these cells. The results suggest that in the miconazole-incubated cells, the drug is not able to alter the level of ergosterol of inhibit ergosterol biosynthesis during 10 min incubation and therefore the interaction of the drug neither leads to significant disorganization of membrane components, nor affects permease activity, whereas in the miconazole-grown cells there is ergosterol depletion leading to accumulation of biosynthetic intermediates, resulting in membrane rearrangement thereby causing a major fluidity change. This fluidity change may explain the drastic reduction of amino acid transport in miconazole-grown cells. To investigate whether the reduction in amino acid transport was only due to change in membrane fluidity, as a consequence of absence of ergosterol, the PM-ATPase activity was checked in the miconazole-grown cells and was found to be unaffected, which suggests that changes in amino acid uptake were due to change in membrane fluidity.