Irreversible Membrane Damage Research Articles

A study of spectrin and lipid peroxidation of red blood cell membrane in thalassaemia carrier.

The aim of the present work is to understand the lipid peroxidation of RBC membrane and the spectrin protein content of RBC membrane cytoskeleton of thalassaemic carrier state (trait) of β and hemoglobin E variant (HbE). We have measured the hemoglobin (Hb), malondialdehyde (MDA) and spectrin content of RBC membrane of thalassaemic carrier. The spectrin content (α and β band) of both β and HbE carrier was not changed than normal individuals. However, lipid peroxidation of RBC membrane was significantly increased in both β and HbE trait, and Hb level was also decreased in thalassaemic carrier. It may be assumed that oxidative damage by excess lipid peroxidation may have no role on irreversible membrane damage in β thalassaemia and HbE thalassaemia carrier.

Effect of glucose deprivation and acute glutamate exposure in cultured retinal cells.

The relationship between bioenergetics and the glutamate system was analyzed in a neuronal model of retinal cells in culture, submitted to glucose deprivation and exposed to glutamate for 2 h, and compared with exposure to glutamate in the presence of glucose. Under glucose deprivation, a reduction (about 1.1-fold) in the energy charge of the cells occurred, probably as a result of a decrement (by about 75%) in the cellular redox efficacy, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. In the absence of glucose, exposure of retinal cells to 10 microM glutamate potentiated the reduction in the energy charge (by about 1.2-fold) and induced a significant increase in the uptake of 45Ca2+ by the cells (1.3-fold), although no significant changes were observed in the presence of glucose. Under glucose deprivation, 100 microM glutamate caused an irreversible cell membrane damage, as shown by the significant increase in lactate dehydrogenase (LDH) leakage (about 1.8-fold). A significant increase in membrane depolarization, measured by the reduction of [3H]tetraphenylphosphonium+ ([3H]TPP+) uptake, was also observed after glutamate exposure in the absence of glucose. In the presence of glucose, high glutamate concentrations (10 mM) induced a major increase in Ca2+ entry into the cells and membrane depolarization, without affecting the energy charge or cell survival. In contrast, in the absence of glucose, 10 mM glutamate did not alter Ca2+ accumulation by the cells and a smaller decrease in membrane potential occurred, as compared to 100 microM glutamate exposure. Data shown in this study suggest that during a prolonged (2 h) and acute exposure to high glutamate (10 mM), under glucose deprivation conditions, the neuronal systems have "adaptive" mechanisms that allow the survival of cells. These findings may have implications in neuronal degeneration occurring during brain ischemia.

Role of cytokines in myocardial ischemia and reperfusion

Mediators of myocardial inflammation, predominantly cytokines, have for many years been implicated in the healing processes after infarction. In recent years, however, more attention has been paid to the possibility that the inflammation may result in deleterious complications for myocardial infarction. The proinflammatory cytokines may mediate myocardial dysfunction associated with myocardial infarction, severe congestive heart failure, and sepsis. A growing body of literature suggests that inflammatory mediators could play a crucial role in ischemia–reperfusion injury. Furthermore, ischemia–reperfusion not only results in the local transcriptional and translational upregulation of cytokines but also leads to tissue infiltration by inflammatory cells. These inflammatory cells are a ready source of a variety of cytokines which could be lethal for the cardiomyocytes. At the cellular level it has been shown that hypoxia causes a series of well documented changes in cardiomyocytes that includes loss of contractility, changes in lipid metabolism and subsequent irreversible cell membrane damage leading to cell death. For instance, hypoxic cardiomyocytes produce interleukin-6 (IL-6) which could contribute to the myocardial dysfunction observed in ischemia reperfusion injury. Ischemia followed by reperfusion induces a number of other multi-potent cytokines, such as IL-1, IL-8, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) as well as an angiogenic cytokine/ growth factor, vascular endothelial growth factor (VEGF), in the heart. Intrestingly, these multipotent cytokines (e.g. TNF-α) may induce an adaptive cytoprotective response in the reperfused myocardium. In this review, we have included a number of cytokines that may contribute to ventricular dysfunction and/or to the cytoprotective and adaptive changes in the reperfused heart.

The role of calcium in UVB-induced damage in irradiated ocular lenses.

The purpose of this study was to evaluate the role of altered calcium homeostasis in the development of irreversible membrane damage in the UVB-irradiated ocular lens. In particular, experiments were designed to determine whether restricting calcium influx could prevent membrane damage that typically leads to ion imbalances and lens opacification following short-term exposure to ultraviolet light (UVB). The influx of calcium was reduced by culturing lenses in a low-calcium culture medium containing 0.3 mM Ca2+ rather than physiological concentrations of 1.6 mM. This low-calcium protocol retarded calcium accumulation in UVB-irradiated lenses for 2 days of culture, and opacification was delayed by 24 h. Loss of transparency did occur during the second day of culture, but more slowly than in irradiated lenses cultured in normal-calcium medium. Membrane damage was assessed by evaluating loss in cation transport activity, assessed by measuring 86Rb uptake into cultured lenses. Uptake was markedly inhibited in UVB-irradiated lenses and low-calcium culture did not prevent this inhibition of cation transport, a finding that explains why low-calcium protocol did not help maintain sodium homeostasis in irradiated lenses. Inhibition of cation transport and sodium accumulation eventually caused lens hydration and light scattering during extended culture in the absence of significant calcium elevation. Additional experiments were done to establish whether initial damage sustained by membranes could be repaired through the biosynthesis of new membrane proteins. Incorporation of 14C-histidine in membranes of the UVB-exposed lens was measured to assess membrane synthesis essential for repairing membrane damage. The rate of membrane protein synthesis, assessed by measuring incorporation of labeled amino acids, declined in UVB cataract, despite the prevention of calcium accumulation. These results suggest that one explanation for irreversible gain in sodium and calcium content accompanying opacification is the inability of lenses to replenish damaged membrane proteins comprising ion channels or transporters.

The hypersensitive reaction, membrane damage and accumulation of autofluorescent phenolics in lettuce cells challenged by Bremia lactucae

SummaryThe expression of resistance to Bremia lactucae determined by the resistance genes Dm5/8 and Dm7 in lettuce was examined; incompatibility involved the hypersensitive reaction (HR) which occurred only within penetrated cells at early and late stages of fungal development, respectively. Autofluorescence observed under UV and blue light excitation in cells undergoing the HR was associated with the accumulation of ester‐linked syringaldehyde and caffeic acid on plant cell walls. Two phases of phenolic deposition were identified. The first was highly localized around penetration points and occurred during incompatible and compatible interactions. The second and major phase was only activated after the occurrence of irreversible membrane damage in the penetrated cell and was reduced by inhibitors of mRNA synthesis. Fungal structures, primary and secondary vesicles, intercellular hyphae and haustoria also became autofluorescent during incompatible interactions. Changes in the fluorescence due to preformed phenolics located in the plant cell vacuole were found just before plasma membrane damage became irreversible during the HR. In addition to localized deposition of phenolics, increases in the concentrations of the major free phenolic esters identified as dicaffeoyl tartaric and chlorogenic acids also occurred during incompatible interactions. The results suggest that membrane damage in penetrated cells occurs at different rates in resistance controlled by Dm5/8 and Dm7 and indicate an important role for irreversible membrane damage in lettuce as a key signalling event leading to widespread activation of defence responses in surrounding cells.

Hypoxic stress induces cardiac myocyte-derived interleukin-6.

Hypoxic and ischemic stresses cause a series of well-documented changes in myocardial cells and tissues, including loss of contractility, changes in lipid and fatty acid metabolism, and irreversible membrane damage leading to eventual cellular death. Activated neutrophils are considered to be involved in this myocardial cellular injury. By stimulation of the neutrophils with chemotactic factors, canine neutrophils can be induced to adhere to isolated cardiac myocytes only if the myocytes have been previously exposed to cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1, and IL-6. To examine the possible involvement of IL-6 in ischemia-reperfusion injury, we used cultured rat neonatal cardiac myocytes to study the effects of hypoxic stress on the production of IL-6 by cardiac myocytes. Unstimulated cardiac myocytes (3 x 10(5) cells per dish) produced 320 pg IL-6 over 4 hours in vitro (ie, biological activity equal to 320 pg recombinant IL-6, as detected by bioassay using the MH-60.BSF2 cell line). The incubation of cardiac myocytes under hypoxic conditions for 4 hours induced significantly increased production of IL-6 compared with normoxic conditions (2.82 +/- 0.49 versus 1.64 +/- 0.18 U/mL, P < .05). Furthermore, reoxygenation for 2 hours after 2 hours of hypoxic stress significantly augmented the production of IL-6 by cardiac myocytes (4.34 +/- 0.52 U/mL, P < .05). These responses to hypoxia and reoxygenation were not observed in fibroblasts isolated from the same tissue. Although unstimulated cardiac myocytes lacked IL-6 mRNA expression detectable by Northern blot analysis, hypoxic stress induced the expression of IL-6 mRNA in the cardiac myocytes. Several pathophysiologically relevant factors also augmented IL-6 release from cultured cardiac myocytes, including IL-1 beta, ionomycin, and epinephrine. Cardiac myocytes respond to hypoxic stress to augment the production of IL-6, and the IL-6 derived from cardiac myocytes may play an important role in the progression of myocardial dysfunction observed in cardiac ischemia-reperfusion injury.

Carbon tetrachloride-induced cell death in perfused livers from phenobarbital-pretreated rats under hypoxic conditions and various ionic milieu: Further evidence for calcium-dependent irreversible changes

The role of Ca 2+ in the initiation of carbon tetrachloride (CCl 4) hepatotoxicity was studied using perfused livers isolated from phenobarbital-pretreated rats in a single-pass system. Krebs-Henseleit bicarbonate buffer containing 1.3 mM CaCl 2 (KHB) was the regular ionic milieu. In the liver perfused with fructose-supplemented regular KHB equilibrated with 95% N 2−5% CO 2, infusion of 0.5 mM CCl 4 caused an early uptake of Ca 2+ coupled with K + leakage and Na + uptake within the infusion time of 30 min, which was followed by a marked lactic dehydrogenase (LDH) leakage into the effluent perfusate and further Ca 2+ uptake by the liver. With Ca 2+-free medium, the prenecrotic K + leakage and the successive LDH leakage were suppressed markedly. However, a perfusate exchange from regular to Ca 2+-free KHB at the end of the prenecrotic stage did not protect against the LDH leakage, and the perfusate exchange conversely did not produce LDH leakage. Perfusion of the liver with high K +(Cl −) medium under 20% O 2 markedly suppressed CCl 4-induced LDH leakage even in the presence of Ca 2+, whereas once CCl 4 had acted under regular KHB perfusion, changing the medium to high K + did not further prevent the LDH leakage. High K +-lactobionic acid medium containing Ca 2+ and supplemented with fructose also suppressed LDH leakage under 95% N 2 without the accompanying prenecrotic Ca 2+ uptake. However, a change of the medium after CCl 4 infusion to regular KHB containing Ca 2+ caused LDH leakage and K + leakage, with Ca 2+ uptake. The prevention of LDH leakage in a different ionic milieu may not be due to suppression of CCl 4 bioactivation, since the liver cytochrome P450 content decreased to a similar extent. These findings suggest that entry of extracellular Ca 2+ into hepatocytes coupled with K + leakage and Na + entry is a prerequisite for CCl 4-induced hepatocyte death and that association of Ca 2+ with a CCl 4-derived radical-mediated process may be necessary for early and irreversible plasma membrane damage.

Loss of asymmetric distribution of sarcolemmal phosphatidylethanolamine during simulated ischemia in the isolated neonatal rat cardiomyocyte.

In the present study we have investigated the reorganization of the sarcolemmal phospholipids during the first 60 minutes of simulated ischemia ("ischemia") as induced by anoxia, volume restriction, and nutrient deprivation. Experiments were carried out on [3H]acetate-labeled neonatal rat cardiomyocytes and isolated (nonradiolabeled) sarcolemmal membranes obtained from the same culture system. After 60 minutes of "ischemia," cellular high-energy phosphate (ATP) levels had decreased to approximately 40% of the control values, but no significant phospholipid hydrolysis was detected. Labeling experiments using the nonpermeant (primary amine-containing phospholipid) probe trinitrobenzenesulfonic acid and nonlytic treatment with (different) exogenous phospholipases A2 were both indicative of a shifted transbilayer distribution of the hexagonalII phase-preferring and fusion-promoting sarcolemmal phosphatidylethanolamine in favor of the outer membrane leaflet. This specific change in sarcolemmal phospholipid asymmetry preceded the loss of integrity of the sarcolemma, monitored by the release of lactate dehydrogenase as well as by scanning electron microscopy. It is proposed that, in addition to the previously reported lateral phospholipid reorganization, uncontrolled transbilayer movement of the non-bilayer-preferring phosphatidylethanolamine from the inner to the outer leaflet of the sarcolemma is an additional factor in destabilizing the lipid bilayer, eventually leading to the irreversible membrane damage seen after a prolonged period of ischemia.

Induction and nuclear accumulation of fos and jun proto-oncogenes in hypoxic cardiac myocytes

Hypoxic and ischemic stresses cause a series of well documented changes in myocardial cells and tissues, including increased anaerobic glycolysis, loss of contractility, changes in lipid and fatty acid metabolism, and eventual irreversible membrane damage and cell death. In this article we describe changes in the expression and regulation of the proto-oncogenes fos and jun in cardiac myocytes exposed to severe hypoxia. The mRNAs encoding c-Fos, c-Jun, Jun-D, and Jun-B were induced within 1 h of exposure to hypoxia, increased 5-10-fold between 1 and 4 h and then declined. These inductions coincided with loss in myocyte contractility but occurred before there was irreversible cell damage or significant ATP loss. Immunostaining with anti-Fos and anti-Jun antibodies revealed the accumulation of these proteins in hypoxic cell nuclei. Pre-treatment of cells with protein kinase inhibitors significantly repressed the response at the mRNA level. We propose that hypoxic stress in these cells activates signal transduction pathways, possibly involving protein kinases, that result in the inductions of fos and jun gene families. Therefore AP1 may regulate myocardial adaptive responses to hypoxia in advance of energy depletion, cell damage, or reoxygenation.

Expression and function of HLA‐B27 in lipid‐linked form: Implications for cytotoxic T lymphocyte‐induced apoptosis signal transduction

Major histocompatibility complex (MHC) class I antigen-restricted cytotoxic T lymphocytes (CTL) kill their target cells not only by inducing irreversible membrane damage but also by triggering a programmed suicide cascade (apoptosis) in target cells. Recent evidence suggests that MHC class I antigens are involved in apoptosis signal transduction in T cells. Therefore, it is possible that MHC class I antigens are also responsible for CTL-induced signal transduction in target cells leading to apoptosis. To test this hypothesis, we have expressed HLA-B27 in Chinese hamster ovary (CHO) cells in a phosphatidyl inositol (PI) anchored form. The expressed Pl-anchored HLA-B27 (PI-B27), a 42-kDa molecule which can be cleaved off the cell surface by PI-specific phospholipase C, can function as an MHC restriction and antigen presentation element for specific CTL. Furthermore, PI-B27 transfectant CHO cells undergo rapid DNA fragmentation when pulsed with the appropriate peptide and treated with specific CTL, suggesting that the cytoplasmic and transmembrane domains of the heavy chain of class I MHC molecules are not required in CTL-induced apoptosis signal transduction in target cells.

A model for compression trauma: pressure-induced injury in cell cultures.

An increase in pressure up to 15 atm was used to condense the cellular membrane of cells in culture thereby eliciting a mechanical-like trauma. This trauma is similar to a compression-like spinal cord injury or brain injury. The cells used in this study were ROC-1 oligodendroglia, N1E-115 neuroblastoma, and human umbilical vein endothelial (HUVE) cells. Total fatty acid (FA) release and release of lactate dehydrogenase (LDH) into the extracellular medium were used as indices of cellular trauma. Pressure-induced FA release, dependent on pressure and pressure duration, occurred with all cell types. The level of pressure needed to cause the greatest increase in FA levels was 10 atm for ROC-1 cells (3 min duration), 15 atm for N1E-115 cells (3 min duration), and 15 atm for HUVE cells (10 min duration). With each cell type, the released FA were reacylated or metabolized between 10 and 30 min of recovery. Following a 12- to 24-h recovery period, N1E-115 and HUVE cells release more FA, indicating that the initial perturbation of the membrane was not fully reversible. LDH levels were significantly increased in both the N1E-115 and HUVE cultures following 24 h of recovery. This efflux of LDH indicates irreversible membrane damage, suggesting that the trauma may be irreversible at longer recovery times.

Ischemic changes in myocardial ionic contents of the isolated perfused rat hearts as studied by NMR.

Using 31P-, 23Na- and 39K-NMR, we assessed ischemic changes in high energy phosphates and ion contents of isolated perfused rat hearts continuously and systematically. To discriminate intra- and extracellular Na+, a shift reagent (Dy(TTHA)3-) was used in 23Na-NMR study. In 39K-NMR study, the extracellular K+ signal was suppressed by inversion recovery pulse sequence in order to obtain intracellular K+ signal without using shift reagents. During the early period of ischemia, increases in intracellular Na+ and inorganic phosphate (Pi) were observed in addition to the well-documented decreases in creatine phosphate and ATP and a fall of intracellular pH, suggesting an augmented operation of Na(+)-H+ exchange triggered by a fall of the intracellular pH resulted from breakdown of ATP. At around 15 min of ischemia, a second larger increase in intracellular Na+ and a decrease in intracellular K+ were observed in association with a second increase in Pi. This was accompanied by an abrupt rise of the ventricular end-diastolic pressure. As there was a depletion of ATP at this time, the increase in intracellular Na+ and associated decrease in intracellular K+ may be explained by inhibition of the Na(+)-K+ ATPase due to the depletion of ATP. A longer observation with 31P-NMR revealed a second phosphate peak (at lower magnetic field to ordinary Pi peak) which increased its intensity as ischemic time lengthened. The pH of this 2nd peak changed in parallel with the changes in pH of the bathing solution, indicating the appearance of a compartment whose hydrogen concentration is in equilibrium with that of the external compartment. Thus, the peak could be used as an index of irreversible membrane damage of the myocardium.

Comparative study of interactions between Bremia lactucae and lettuce cells in suspension culture and in cotyledons

A critical comparison of the relationship between fungal development and irreversible membrane damage (IMD) in lettuce cells penetrated by Bremia lactucae in suspension cultures or in the epidermis of the cotyledon petiole has revealed the following important differences between interactions occurring in vitro and in the plant. (1) Large fungal colonies comprising intercellular hyphae and the formation of numerous haustoria did not develop in suspension cultures. (2) IMD occurred at earlier stages of fungal development in cultured cells than in cotyledons of susceptible cultivars. (3) In resistant cells undergoing the hypersensitive reaction (HR) fungal development was more advanced in vitro than in the plant at the time of IMD. (4) Suppression of the plant's metabolic activity either by heat-shock or blasticidin S (an inhibitor of protein synthesis) had less effect on the HR in cultured cells than in the plant. Despite these differences, B. lactucae did produce characteristic infection structures in cultured cells and, in certain cultivar/isolate interactions e.g. cvs. Diana ( Dm 1 + Dm 3 + Dm 7 + Dm 5/8) and Cobham Green (no known resistance genes) with isolates V0/11 (with at least one dominant avirulence allele at loci A 1, A 3, A 7 and A 5/8) and TV (with no dominant avirulence alleles at loci A 1, A 3, A 7 or A 5/8), the expected compatibility or incompatibility was clearly expressed. By contrast, the expected rapid HR of cells of cv. Valmaine ( Dm 5/8) challenged by isolate V0/11 was not observed in cultured cells. The influence of nutrient status on the responses of cultured lettuce cells is discussed.

A promising approach for improving the recovery of heart transplants: Prevention of free radical injury through iron chelation by deferoxamine

Iron catalysis is involved in the generation of the highly cytotoxic hydroxyl radical and in the chain reactions of subsequent lipid peroxidation that lead to irreversible membrane damage. Assuming that ischemically stored heart transplants may incur free radical injury at the time of reoxygenation, we assessed the effects of the iron chelator deferoxamine in 70 isolated isovolumic buffer-perfused rat hearts subjected to the following protocol: cardioplegic arrest; cold (2 degrees C) storage for 5 hours; global ischemia at 15 degrees C for 1 hour, intended to simulate the implantation procedure; and normothermic reperfusion for 1 additional hour. During poststorage ischemic arrest, the following techniques of myocardial protection were evaluated: hypothermia alone; high-pressure (60 cm H2O) cardioplegia given at 0, 30, and 55 minutes of arrest; low-pressure (30 cm H2O) cardioplegia given at 0 and 55 minutes of arrest; and low-pressure (30 cm H2O) cardioplegia only given at 55 minutes of arrest. Treated hearts had deferoxamine (6 mumol) added to the cardioplegic solution used throughout the experimental time course. Further, in the treated group subjected to the protocol of single cardioplegic delivery at end ischemia, deferoxamine was given both in the cardioplegic reperfusate and in the Krebs buffer over the 15 initial minutes of reflow. Based on comparisons of postreperfusion ventricular pressure development, maximal rate of rise of ventricular pressure, left ventricular compliance, and coronary flow, the best myocardial protection was afforded by deferoxamine given as an additive to single-dose cardioplegic solution at the end of arrest and to the reperfusate during the initial phase of reoxygenation. As the drug has no inotropic effect, its protective action is most likely related to a decrease in catalytic iron available for free radical production and lipid peroxidation. These results support the hypothesis that oxidative damage may contribute to donor heart failure and demonstrate that this form of damage can be efficiently acted upon by iron chelation. The clinical relevance of these data stems from the fact that deferoxamine is available for human use and might become an effective means of improving donor heart preservation in the setting of clinical heart transplantation.

Effects of heat-shock and inhibitors of protein synthesis on irreversible membrane damage occurring during the hypersensitive reaction of Lactuca sativa L. to Bremia lactucae Regel

The effect of heat-shock on the irreversible membrane damage (IMD) which occurs during the hypersensitive reaction of lettuce cultivars resistant to downy mildew disease was investigated. Combinations of cultivars and fungal isolates were used which allowed expression of the Dm1, Dm3 and Dm5/8 genes for resistance in hosts with different genetic backgrounds. In all plant genotypes, heat-shock (exposure to 55 °C for 45 s) 0·5 h before inoculation delayed IMD at most infection sites, at least until the development of intracellular hyphae within penetrated cells. Without heatshock, IMD occurred during expansion of the fungal primary vesicle in cultivars expressing resistance gene Dm5/8 or during expansion of the secondary vesicle with Dm1 and Dm3. In Dm1: A1 and Dm3: A3 interactions, heat-shock applied 17 h before inoculation did not delay the occurrence of IMD. By contrast, in Dm5/8: A5/8 interactions, a 17 h delay between heat-shock and inoculation did not reduce the suppression of IMD. Heat-shock 0·5 h before inoculation also led to an increase in sporulation during incompatible interactions involving the Dm5/8 gene. The effect on sporulation was reduced by increasing the delay between treatment and inoculation. Thus, with isolate V0/11 in cv. Valmaine ( Dm5/8) sporulation occurred when inoculation was delayed for 1 or 2 days, but if delay was for 3 days no conidiosporangia were produced. Treatment with the translation inhibitor blasticidin S (12·5 μg ml −1) delayed the time of IMD in cvs Valmaine ( Dm5/8) and Dandie ( Dm3) following inoculation with isolate V0/11 ( A1–8). However, the inhibitors of transcription, actinomycin D and cordycepin, at concentrations up to 180 μg ml −1, caused little if any delay in the onset of irreversible damage to the host plasma membrane. Results suggest that de novo protein synthesis, but not mRNA synthesis, is required for the occurrence of IMD during the HR in lettuce, and that heat-shock differentially affects expression of genes for resistance to downy mildew disease.

Fungal development and irreversible membrane damage in cells of Lactuca sativa undergoing the hypersensitive reaction to the downy mildew fungus Bremia lactucae

A plasmolytic method was developed to examine irreversible membrane damage (IMD) occurring during the early stages of the hypersensitive reaction (HR) of lettuce ( Lactuca sativa L.) to Bremia lactucae Regel. Symptoms associated with incompatibility determined by Dm genes 1–8 in lettuce were compared. The timing of IMD varied according to the Dm gene expressed and occurred within 1·5 h of penetration during the expansion of the primary vesicle in the most rapid reactions which were determined by Dm 2 and Dm 5/8. By contrast, with Dm 1 or Dm 3, IMD occurred only after initiation of the secondary vesicle, from 3 h after penetration, and rarely occurred during the first 12 h after inoculation of genotypes possessing Dm 4, Dm 6 or Dm 7. The genetic background of the host had little effect on the expression of Dm genes 1, 3 or 5/8, but some fungal isolates, thought to have the same gene for avirulence, differed in their behaviour on resistant cultivars. The time of occurrence of IMD in the first 12 h after inoculation had a direct bearing on the extent of fungal development during the subsequent 36 h. In lettuce cv. Valmaine ( Dm 5/8 gene for resistance) growth of the avirulent isolate VO/11 of B. lactucae was checked at the time of IMD. In all the combinations of isolates and cultivars studied, plasmolysis demonstrated lack of adhesion between primary and secondary vesicles and the host plasmalemma during compatible or incompatible interactions. Variation existed between isolates in their speed of germination and initiation of penetration on different varieties, but variation was not related to the expression of Dm genes.

Metabolic depletion preceding cytolysis induced by antibody and complement as revealed by 31P-NMR spectroscopy.

The mechanism whereby fixation of the membrane attack complex of complement (C') induces irreversible membrane damage ultimately leading to cell death may not be an exclusive membrane event. We investigated possible involvement of cellular metabolism in the lysis of nucleated cells induced 3by antibody (Ab) against cell surface antigens and C'. Using 31P-NMR spectroscopy we observed a marked reduction of creatine phosphate and relatively unaltered ATP levels shortly after the application of C', but prior to the onset of lysis. These metabolic changes preceded uptake of vital stairlT (e.g., eosine), or release of isotopically [\left( N{{a}_{2}}^{51}Cr{{O}_{4}} \right)\] -labeled cytoplasmic components. Monitoring the course of natural cell death over a 6-hr period revealed a comparable depletion of creatine phosphate prior to cell death. We would like to propose that membrane fixation of the C' attack complex induce metabolic depletion of high energy components which in turn cause irreversible cell swelling and membrane damage leading to cytolysis.

Glycerol-induced baroprotection in erythrocyte membranes

A system for applying hydrostatic pressures up to 10,000 atm upon cell suspensions for time intervals from a few seconds to several minutes is described. The K − content of toad red blood cells was used as an indication of the degree of membrane injury induced by the hyperbaric condition. It is practically not affected for pressures up to 2000 atm in experiments lasting 3 or 10 min, falling markedly for pressures of 5000 or 8000 atm. The duration of the applied pressure and its intensity are additive regarding the magnitude of the baroinjury. Glycerol, a cryoprotective agent, at 4.0 M, confers partial but significant baroprotection. which is characterized by a smaller decline of the cell K − content of the glycerol-treated cells in comparison to the untreated cells, submitted to the same conditions of pressure and time. Baroinjury is compatible with a reversible mechanism. However, irreversible membrane damage occurs for a pressure of 8000 atm applied for 10 min. Baroinjury is discussed in terms of alterations of the lipid leaflet or of membrane proteins, and the mechanism of baroprotection in terms of stabilization of membrane components, under the effect of high pressure, by the association of glycerol with the proteins or the phosphate head groups of phospholipids.

ATP involvement in plant tissue responses to low temperature

The effects of chilling and freezing temperature on membrane permeability and ATP content were studied in the leaves of cucumber (Cucumis sativus L.) and winter rape (Brassica napus L. var. oleifera L.) leaves, grown at different temperatures. In the winter rape leaves, the endogenous ATP content was modified by application of dinitrophenol (DNP) solutions of different concentrations. The low temperature‐induced changes in membrane permeability (as monitored by the conductivity method) were found to be associated with ATP decrease, both in the chilling‐sensitive and chilling‐resistant (subjected to freezing) plants. In tissues showing reversible injuries, changes in ATP content preceded those in membrane permeability and the adenylate energy charge was affected slightly. In tissues showing irreversible membrane damage, the ATP content was always below 0.4 μmol (g dry weight)−1 and the adenylate energy charge was near 0.5. DNP treatment increased freezing sensitivity of winter rape leaves. In the cold‐hardened winter rape leaves, however, freezing and thawing did not significantly affect ATP content or the energy charge, although the specimen showed a rather large increase in membrane permeability. In these leaves ATP content recovered about 20 h after a freezing and thawing treatment. It is proposed that a decrease in ATP supply might be the primary reason for the membrane leakiness at low temperature, both in chilling‐sensitive and chilling‐resistant (subjected to freezing) plants. The conclusion is, however, not true for the cold‐acclimated, frostadapted cells.

Thylakoid membrane stability in drought-tolerant and drought-sensitive plants

The stress stability of membranes from two drought-tolerant plants (Craterostigma plantagineum andCeterach officinarum) was compared with that of a drought-sensitive plant (Spinacia oleracea) in model experiments. Thylakoids from these plants were exposed to excessive sugar or salt concentrations or to freezing. All stresses caused loss of membrane function as indicated by the loss of cyclic photophosphorylation or the inability of the membranes to maintain a large proton gradient in the light. However, loss of membrane functions caused by osmotic dehydration in the presence of sugars was reversible. Irreversible membrane damage during freezing or exposure to salt was attributed mainly to chaotropic solute effects. The sensitivity to different stresses was comparable in thylakoid membranes from tolerant and sensitive plants indicating that the stress tolerance of a plant can hardly be attributed to specific membrane structures which would increase membrane stability. Levels of membrane-compatible solutes such as sugars or amino acids, among them proline, were much higher in the drought-tolerant plants than in spinach. Isolated thylakoids suspended in solutions containing an excess of sugars remained functional after dehydration by freeze-drying. This indicates that membrane-compatible solutes are important in preventing membrane damage during dehydration of poikilohydric plants.