Nystatin and killer toxin sensitivity of free and immobilizedSaccharomyces cerevisiae

The inhibitory effect of nystatin and killer toxin on the growth of free and covalently-immobilizedSaccharomyces cerevisiae cells was studied. The resistance of immobilized cells to both agents was accompanied by increased amounts of phospholipids and sterols. The possible relationship between these changes in the membrane composition and the transduction of a signal across the cytoplasmic membrane is discussed.     

Cholesterol incorporation intoBacillus megaterium without compositional modification of membrane lipids in response to changes of the membrane functions

Cholesterol was incorporated intoBacillus megaterium, resulting in reduction of membrane permeability and fluidity. However, an apparent modification of membrane composition was found neither in fatty acids nor in phospholipids, whereas electrophoretic patterns of the membrane proteins were changed. Unlike eukaryotic and mycoplasma cells, cholesterol incorporation intoB. megaterium influences the membrane functions but is not accompanied by compensatory changes in the membrane lipids, which appear to be a response common to wallcovered and cholesterol-nonrequiring bacteria.     

Structural changes ofNoetia ponderosa red blood cell membranes during cell volume regulation in reduced salinities: A freeze fracture study

Summary Changes in molluscan blood cell membrane structure coincided with changes in membrane amino acid permeability during cell volume regulation. Blood cells were freeze fractured after the free amino acid permeability of their membranes had been altered by modifying the extracellular Ca²⁺ and intracellular ATP levels and the membrane particles examined for changes in size, number/area and distribution. Test substances that altered the divalent cation or ATP levels also altered membrane particle densities, but not size or distribution, of freeze fractured blood cells. Those test substances (Ca²⁺-free seawater, DNP, low temperature) that inhibited volume regulation and the FAA efflux caused decreased membrane particle density, while those test substances (Co²⁺, Mn²⁺) that potentiated volume regulation and the FAA efflux increased the number of membrane particles/unit area. These changes in membrane particle density appear to result from the changes in surface area due to the treatment effects on cell volume, so that the number of membrane particles per cell remained constant. Therefore, altered membrane FAA permeability is associated with altered membrane particle density, but the effect of this structural alteration on membrane permeability is not clear.Abbreviations FAA free amino acid - DMSO dimethylsulfoxide - DNP dinitrophenol - ASW artificial seawater     

Effect ofcis andtrans unsaturated fatty acids on the transport properties ofSalmonella typhimurium

The transport of α-methyl-D-glucoside and two aminoacids, L-phenylalanine and L-leucine by a temperature sensitive fatty acid requiring mutant ofSalmonella typhimurium was studied under conditions of supplementation withcis or trans-unsaturated fatty acids. The results of such experiments definitely establish a relationship between the fatty acids composition of the membrane and the transport property of the cells. Cells grown in the presence of trans-unsaturated fatty acids cannot transport so efficiently as compared to the cis-unsaturated fatty acid-grown cells except linolelaidic acid, atrans-trans-unsaturated fatty acid. Protein: phospholipid ratio of the membrane also varies significantly under such conditions. The affinity of L-phenylalanine transport carrier for the substrate changes remarkably in cells grown in the presence of differentcis or trans-unsaturated fatty acids and indicate the possible role of membrane lipids in membrane assembly as well as regulation of the activity of L-phenylalanine transport system.     

Long- and short-term freezing induce different types of injury in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaf cells

In nature, intact plant cells are subjected to freezing and can remain frozen for prolonged periods. We assayed the survival of Arabidopsis thaliana leaf cells following freezing and found that short- and long-term exposures produced different types of cellular injury. To identify the cause of these injuries, we examined the ultrastructure of the cell plasma membranes. Our results demonstrate that ultrastructural changes in the plasma membrane due to short-term freezing are associated with interbilayer events, including close apposition of the membranes. In both acclimated and non-acclimated leaf cells, these interbilayer events resulted in “fracture-jump lesions” in the plasma membrane. On the other hand, long-term freezing was associated with the development of extensive protein-free areas caused by the aggregation of intramembrane proteins with consequent vesiculation of the affected membrane regions; this effect was clearly different from the ultrastructural changes induced by interbilayer events. We also found that prolonged exposure of non-acclimated leaf cells to a concentrated electrolyte solution produced effects that were similar to those caused by long-term freezing, suggesting that the ultrastructural changes observed in the plasma membrane following long-term freezing are produced by exposure of the leaf cells to a concentrated electrolyte solution. This study illustrates multiple causes of freezing-induced injury in plant cells and may provide useful information regarding the functional role of the diverse changes that occur during cold acclimation.     

Drug—induced alterations in the ultrastructural organization of Histoplasma capsulatum and Blastomyces dermatitidis

Aspects of the fine structure as seen in thin section of yeastlike cells ofHistoplasma capsulatum andBlastomyces dermatitidis exposed to polyenic antibiotics are described and illustrated by electron micrographs. The exposure of log phase yeastlike cells to minimal fungicidal concentrations of both amphotericin B (Fungizone) and hamycin resulted in detectable alterations of the plasma membrane, and, to a lesser extent, the mitochondria. WithH. capsulatum, ultrastructural changes were observed to occur within 1 h exposure to amphotericin B. Marked degenerative changes and plasmolysis were observed to occur within 6 hrs exposure of the yeastlike cells to both polyenes. The observed changes in ultrastructural appearance are compatible with the concept of binding of the polyene with membrane sterol and subsequent damage due to alterations of permeability.     

Changes in leaf ultrastructure and carbohydrates inArabidopsis thaliana L. (Heyn) cv. Columbia during rapid cold acclimation

Summary We studied cell ultrastructure and carbohydrate levels in the leaf tissue ofArabidopsis thaliana L. (Heyn) cv. Columbia during rapid cold acclimation. Freezing tolerance of the leaves from 26 day old plants was determined after 48 h and 10 days at 4°C. Acclimation treatment of 48 h decreased the lethal freezing temperature from –5.7°C to –9.4°C. Freezing tolerance was not altered further by acclimation at 4 °C for 10 days. Ultrastructural changes in the parenchyma cells were evident after 6 to 24 h of cold acclimation. The plasma membrane showed signs of extensive turnover. Evidence of membrane invaginations and sequestering of membrane material was observed. In addition, numerous microvesicles, paramural bodies, and fragments of endoplasmic reticulum were noticed in the vicinity of plasma membrane. Modifications in the structure of cell membranes were evident after 5 days of exposure to low temperature. Small, darkly stained globules were seen on the plasma membrane, tonoplast, chloroplast envelope membrane, mitochondrion outer membrane, dictyosome cisternae membrane, and microvesicle membrane. As far as we are aware, this type of membrane modification has not been described previously in plant cells exposed to low temperature. We propose to call these structures membraglobuli. Acclimation treatment also increased the concentrations of soluble sugars and starch. These observations suggest that cold acclimation inA. thaliana induces changes in both plasma membrane properties and carbohydrate composition.     

Alteration of ergosterol content and chitin synthase activity inCandida albicans

Chitin synthase activity in total membrane fractions from two polyene-resistant, ergosterol-deficient mutants ofCandida albicans was significantly higher in comparison to the parental polyene-sensitive strain. The zymogen component from membrane preparations of stationaryphase cells of ergosterol mutants was more susceptible to trypsin digestion than those from the parental polyene-sensitive strain. Mechanisms that might explain the effect of changes in membrane composition in the mutant strains on chitin synthase activity are discussed.     

DIFFERENCES IN PHOTOSYNTHESIS-ASSOCIATED PROPERTIES OF THE BLUEGREEN ALGA SYNECHOCOCCUS CEDRORUM CROWN AT 30 AND 40 C1

The growth of Synechococcus cedrorum Saug. (UTEX 1191) at 40 C resulted in structural and functional alteratons relative to cells grown at 30 C. Structural variations included cell morphology and the chemical composition of the membrane. Growth at 40 C. produced cells that were longer and thinner than those at 30 C. The fatty acid composition changed substantially upon growth at 40 C. yielding a distribution with a higher ratio of: i) saturated to unsaturated fatty acids and; ii) longer chain unsaturated fatty acids. Furthermore, the pattern of membrane proteins as determined by sodium dodecyl sulfate electrophoresis was distinctly different. The functional changes were typified by photosynthetic rates which were approximately half those of the 30 C grown cells. A number of spectral parameters were also seen to change in 40 C. grown cells: absorption spectra indicated a higher phycocyanin : chlorophyll ratio. Low temperature fluorescence spectra were consistent with a lowered efficiency of energy transfer from phycocyanin to chlorophyll. It is suggested that the fatty acid changes at 40 C. yield a more fluid membrane which is responsible for the functional alterations. The modification of phycocyanin. chlorophyll ratios, as well as the appearance of P750, is discussed with respect to membrane fluidity.     

Kinetic Properties of the Channels Formed by the Bacillus thuringiensis Insecticidal Crystal Protein Cry1C in the Plasma Membrane of Sf9 Cells

Spectrofluorimetric measurements were conducted to quantify, in real-time, membrane permeability changes resulting from the treatment of Sf9 insect cells (Spodoptera frugiperda, Lepidoptera) with different Bacillus thuringiensis Cry insecticidal proteins. Coumarin-derived CD222 and Merocyanin-540 probes were respectively used to monitor extracellular K⁺ and membrane potential variations upon Sf9 cells incubation with Cry toxins. Our results establish that Cry1C induces, after a delay, the depolarization of the cell membrane and the full depletion of intracellular K⁺. These changes were not observed upon Sf9 cells treated with Cry1A family toxins. Both the rate of the K⁺ efflux and the delay before its onset were dependent on toxin concentration. Both parameters were sensitive to temperature but only the delay was affected by pH. Cry1C-induced K⁺ efflux was inhibited by lanthanum ions in a dose-dependent manner. This study provides the first kinetic and quantitative characterization of the ion fluxes through the channels formed by a Cry toxin in the plasma membrane of a susceptible insect cell line. Received: 4 October 1999/Revised: 21 December 1999     

Uterine epithelial cell changes during pregnancy in a marsupial (Sminthopsis crassicaudata; Dasyuridae)

Formation of a placenta requires intimate contact between the embryonic and maternal uterine epithelia in early pregnancy. Contact is accompanied by a characteristic suite of changes to the plasma membranes of uterine epithelial cells, termed the plasma membrane transformation. The plasma membrane transformation occurs in eutherian mammals and in viviparous (live‐bearing) squamate reptiles, and may be fundamental to the evolution of viviparity in amniotes. Marsupials provide an excellent opportunity to test the generality of this phenomenon. Here, we present the first detailed study of the plasma membrane transformation in a marsupial. We combine electron microscopy and immunohistochemistry to describe morphological and molecular features of uterine epithelial cells during pregnancy in the fat‐tailed dunnart (Sminthopsis crassicaudata; Dasyuridae). Cell morphology changes dramatically in S. crassicaudata during pregnancy. Apical microvilli are replaced by irregular blunt projections, then by spiky projections postimplantation. Cell surfaces flatten and ciliated cells are lost. Junctional complexes between adjacent cells increase in depth, then decrease just before implantation, which is consistent with junctional protein localization in this region of the cell membrane. The uterine cellular changes in S. crassicaudata are consistent with a plasma membrane transformation, and support the idea that this phenomenon is fundamental to the evolution of viviparity in amniote vertebrates. J. Morphol. 275:1081–1092, 2014. © 2014 Wiley Periodicals, Inc.     

Membrane maintenance and electrical properties of photoreceptors of wild-type andrpa (receptor potential absent) mutant blowflies (Calliphora erythrocephala)

Summary The maintenance of photoreceptor cell membranes in the blowfly was investigated in relation to the diurnal cycle, age, and therpa (receptor potential absent) phototransduction mutation. The effect of disturbed membrane assembly on the electrical membrane properties was examined using single-electrode discontinuous current-clamp techniques. In wild-type flies the cross-sectional dimensions of the rhabdomeres were markedly reduced with age, and the quantity of synthetic organelles decreased concurrently, whereas no correlation was found between the diurnal cycle and membrane turnover. Therpa mutation is thought to block the visual transduction cascade in photoreceptor cells and to lead to degeneration of the photoreceptor cell bodies. The volume of rhabdomeres decreased markedly inrpa mutants and the quantity of synthetic organelles was reduced significantly, indicating an imbalance between photoreceptive membrane renewal and degradation. Also, the plasma membrane underwent degenerative changes. The passive electrical properties of photoreceptor cells — resting membrane voltages and input resistances — were only slightly changed from those of wild-type flies, although the photoreceptive membrane did not depolarize in response to light. This indicates no apparent disturbance in the function of the ionic channels in these membranes. Taken together, these results suggest that the photoreceptor cells need a functional phototransduction cascade with its feedback controls to maintain continuous renewal of rhabdomeres, but that the plasma membrane maintains its normal electrochemical properties despite extreme morphological degeneration of photoreceptor cell.     

Physiological characteristics and regulation mechanisms of the H+ pumps in the plasma membrane and tonoplast of characean cells

The relationship between the physiological characteristics and changes in the activities of H⁺ pumps of the plasma membrane and tonoplast of characean cells is discussed. The large size of the characean internodal cells allows us to perform various experimental operations. The intracellular perfusion technique developed by Tazawaet al. (1976) is a powerful tool for analyzing the characteristics and control mechanisms of the H⁺ pumps (Tazawa and Shimmen 1987, Tazawaet al. 1987, Shimmenet al. 1994) Respiration-dependent changes in the activity of the plasma membrane H⁺ pump are explained by changes in the supply of energy substrate. Photosynthesis-dependent changes in activities of both the plasma membrane and the tonoplast H⁺ pumps are explained in terms of changes in the level of inorganic phosphate in the cytoplasm. Cytoplasmic and vacuolar pHs are suggested to be controlling factors forin vivo activities of the H⁺ pumps. Furthermore, the membrane potential and various ions are considered to bein vivo factors that regulate the activities of the H⁺ pumps. Recipient of the Botanical Society Award of Young Scientists, 1993.     

Transplasmalemma electron transport is changed in simian virus 40 transformed liver cells

Transplasma membrane electron transport activity by fetal rat liver cells (RLA209-15) infected with a temperature-sensitive strain of SV40 has been measured with cells grown at the restrictive temperature (40°C) and permissive temperature (33°C). The transformed cells grown at 33°C had only one-half the rate of external ferricyanide reduction as the nontransformed cells held at 40°C. Both theK _m andV _max for ferricyanide reduction were changed in the transformed state. The change inV _max can be based on a decrease of NADH in the transformed cells. The change in rate with ferricyanide does not depend on change in surface charge. Reduction of external ferricyanide was accompanied by release of protons from the cells. The ratio of protons released to ferricyanide reduced was higher in the transformed cells than in the non-transformed cells. Since the transplasma membrane electron transport has been shown to stimulate cell growth under limiting serum, the changes in the plasma membrane electron transport and proton release in transformed cells may relate to modification of growth control.     

Rates of chilling to 0°C: implications for the survival of microorganisms and relationship with membrane fluidity modifications

The effects of slow chilling (2°C min⁻¹) and rapid chilling (2,000°C min⁻¹) were investigated on the survival and membrane fluidity of Escherichia coli, of Bacillus subtilis, and of Saccharomyces cerevisiae. Cell death was found to be dependent on the physiological state of cell cultures and on the rate of temperature downshift. Slow temperature decrease allowed cell stabilization, whereas the rapid chilling induced an immediate loss of viability of up to more than 90 and 70% for the exponentially growing cells of E. coli and B. subtilis, respectively. To relate the results of viability with changes in membrane physical state, membrane anisotropy variation was monitored during thermal stress using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. No variation in the membrane fluidity of all the three microorganisms was found after the slow chilling. It is interesting to note that fluorescence measurements showed an irreversible rigidification of the membrane of exponentially growing cells of E. coli and B. subtilis after the instantaneous cold shock, which was not observed with S. cerevisiae. This irreversible effect of the rapid cold shock on the membrane correlated well with high rates of cell inactivation. Thus, membrane alteration seems to be the principal cause of the cold shock injury.     

Study of phototrophic purple bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> cell morphology of wild-type and ipt-transformant by atomic force and electron microscopy

A comparative study of phototrophic purple bacterium Rhodobacter sphaeroides cell morphology of wild-type and ipt-transformant was done by atomic force and electron microscopy. It was shown that transformation led to a decrease in the number or total disappearance of the flagella, as well as to changes in the structure of the outer membrane of the bacteria cell wall. On the wild-type cell surface phage-like structures were found, and in transformed cells at their places hollows were identified. This study significantly extends an understanding of the changes occurring in the ipt-transformants of phototrophic purple bacterium Rhodobacter sphaeroides. This investigation not only confirmed earlier obtained data about the differences in the wild-type and ipt-transformant phototrophic purple bacteria cell wall, but also showed fine changes in the structure of its outer membrane.     

Evidence for a correlation between swimming velocity and membrane fluidity of Tetrahymena cells

The influence of the physical state of the membrane on the swimming behaviour of Tetrahymena pyriformis was studied in cells with lipid-modified membranes. When the growth temperature of Tetrahymena cells was increased from 15°C to 34°C or decreased from 39°C to 15°C, their swimming velocity changed gradually in a similar to the adaptive change in membrane lipid composition. Therefore, such adaptive changes in swimming velocity were not observed during short exposures to a different environment. Tetrahymena cells adapted to 34°C swam at 570 μm/s. On incubation at 15°C these cells swam at 100 μm/s. When the temperature was increased to 34°C after a 90-min incubation at 15°C, the initial velocity was immediately recovered. On replacement of tetrahymanol with ergosterol, the swimming velocity of 34°C-grown cells decreased to 210 μm/s, and the cells ceased to move when the temperature was decreased to 15°C. To investigate the influence of the physical state of the membrane on the swimming velocity, total phospholipids were prepared from Tetrahymena cells grown under these different conditions. The fluidities of liposomes of these phospholipid were measured using stearate spin probe. The membrane fluidity of the cells cooled to 15°C increased gradually during incubation at 15°C. On the other hand, the fluidity of the heated cell decreased during incubation at 34°C. Replacement of tetrahymanol with ergosterol decreased the membrane fluidity markedly. Consequently, a good correlation was observed between swimming velocity and membrane fluidity; as the membrane fluidity increased, the swimming velocity increased linearly up to 600 μm/s. These results provide evidence for the regulation of the swimming behaviour by physical properties of the membrane.     

Fluorescent probe studies of normal, persistently infected, rous sarcoma virus-transformed, and trypsinized rat cells

The fluorescent probes pyrene, pyrene butyric acid and N-phenyl 1-naphthylamine were used to study membranes of normal cells, RSV-transformed cells, cells treated with a proteolytic enzyme, and cells persistently infected with lymphocytic choriomeningitis virus. The lifetimes of excited pyrene and pyrene butyric acid showed only minor changes when these probes were in normal, transformed, trypsinized or persistently infected cells. However, pyrene, but not pyrene butyric acid, lifetimes are shorter in cell membranes than in homogeneous solvents. The quenching of excited pyrene in cells by quencher molecules was slower than corresponding reactions in homogeneous solutions indicating that the probe was screened from the quenchers by the membrane. However, quenching reactions with the pyrene butyric acid probe were similar in cells and homogeneous solvents. This indicates that pyrene and pyrene butyric acid reside in different lipid regions of the membrane. Transformed and trypsinized cells showed increased membrane fluidity compared to normal and persistently infected cells. Membrane fluidity was determined from the excimer/monomer fluorescence ratios of pyrene, and by the polarization of N-phenyl 1-naphthylamine fluorescence. Several techniques distinguished between normal and transformed or trypsinized cells; however, the only parameter unique to viral transformation was a blue shift of the fluorescence maxima of N-phenyl 1-naphthylamine. This shift reflected a less polar environment for N-phenyl 1-naphthylamine in virus-transformed cells.     

Exposure of <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis> to Red Light in the Presence of the Light-Activated Antimicrobial Agent Toluidine Blue Decreases Membrane Fluidity

Porphyromonas gingivalis, one of the etiological agents of periodontitis, can be killed by red light in the presence of toluidine blue. The purpose of this study was to determine whether this light-induced killing was accompanied by changes in the fluidity of the organism's cytoplasmic membrane. A suspension of the organism was exposed to red light in the presence of toluidine blue, and the membrane fluidity was monitored spectrofluorimetrically by using the membrane probe trimethylammonium diphenyl hexatriene. The fluidity of the organism's cytoplasmic membrane was found to decrease significantly during lethal photosensitization, and this was accompanied by membrane condensation and vacuolation of the cells. Although changes in membrane fluidity are often attributable to lipid peroxidation, malonaldehyde (a product of lipid peroxidation) was not detectable. The disruption of membrane functions associated with a decreased membrane fluidity may contribute to the bactericidal effect of light-activated toluidine blue. Received: 12 October 2001 / Accepted: 7 December 2001     

Ultrastructural study of the life cycle of <Emphasis Type="Italic">Rickettsia slovaca</Emphasis>, wild and standard type,cultivated in L929 and vero cell lines

Ultrastructural changes induced by Rickettsia slovaca standard type (ST) and wild type (WT) were examined during their life cycle in L929 and Vero cells. R. slovaca invaded the cytoplasm of the host cell by phagocytosis on the 1st d p.i. Rickettsiae adhering to the cytoplasmic membrane were engulfed by cellular extensions and occurred in phagocytic vacuoles. Binary fission of rickettsia was observed. The nuclear chromatin of eukaryotic cells was rearranged and condensed during 3rd and 6th d p.i. Finally, loss of the plasma membrane integrity, destruction of cytoplasm and nucleus resulted in cell lysis. Degeneration of the host cell caused by WT and ST was observed after 4 and 5 d p.i. in L929 cells and after 3 and 6 d p.i. in Vero cells, respectively. WT type was able to penetrate into the nucleus of the host cell and was responsible for dilatation of the perinuclear space and endoplasmic reticulum, causing more pronounced and different cytopathological changes than the ST.