Peroxidation of tobacco membrane lipids by the photosensitizing toxin, cercosporin

Cercosporin, a nonspecific toxin from Cercospora species, is a photosensitizing compound which rapidly kills plant cells in the light. Cell death appears to be due to a cercosporin-mediated peroxidation of membrane lipids. Tobacco leaf discs treated with cercosporin showed a large increase in electrolyte leakage 1 to 2 minutes after irradiation with light. All tobacco protoplasts exposed to cercosporin in the light were damaged within 45 minutes. Chloroform:methanol extracts of toxin-treated suspension cultures gave positive reactions for lipid hydroperoxides in the thiobarbituric acid test. Cercosporin-treated leaf discs emitted high concentrations of ethane 12 to 24 hours after incubation in the light. Cercosporin also oxidized solutions of methyl linolenate as determined by the thiobarbituric acid assay and the emission of ethane. α-Tocopherol had an inhibitory effect on the cercosporin-mediated lipid peroxidation.     

Variation of lipid membrane composition caused by strong bending

Dynamic coupling between the morphology and molecular composition of cellular membranes is crucial for formation of the intracellular organelles and transport vesicles. Most of the membrane proteins and lipids discriminate membrane curvatures. However, it remains unclear whether the curvature alone is sufficient to support heterogeneous distribution of lipids. Here we demonstrate that the curvature-driven redistribution of phospholipids, such as dioleoylphosphatidylethanolamine (DOPE), requires strong membrane bending. We used cylindrical lipid nanotubes (NTs) pulled from planar lipid membranes with lateral tension of ∼1 dyn/cm. Such high tensions forced extreme curvatures of the NT membrane, with luminal radius approaching the thickness of the lipid bilayer, 5nm. When the NT contained lipid species with high spontaneous curvature (SC), such as DOPE, we observed slow reduction of its radius. This reduction indicated the redistribution of DOPE between the inner and outer monolayers of the NT. Accordingly, the SC of DOPE was recovered from the measured changes in the radii: the SC value, calculated under the assumption that the DOPE content is coupled to the monolayer curvature, was ∼0.4 nm⁻¹, consistent with the published data. Thus, redistribution of lipids should be taken into account in calculations of composition and material properties of strongly deformed membrane structures, such as intermediate structures arising in the processes of membrane fusion and fission.     

Changes in the structure, composition and function of sarcoplasmic-reticulum membrane during development.

The structure, chemical composition and function of the microsomal fraction, isolated by differential centrifugation and purified on sucrose gradients, from muscle of fetal, newborn and young rabbits were characterized and compared with those of sarcoplasmic reticulum vesicles from adult muscle. Negative staining shows that the microsomal vesicles isolated from muscles of embryos and newborn animals are smooth, in contrast to vesicles obtained from adult muscle which contain 4-nm particles on their surface. The particles appear first in the microsomal vesicles from muscles of 5--8-day-old rabbits. Their number increases with the age of the animals. Ca2+-pump protein, with molecular weight about 100000, accounts for 10% of the total protein content in sarcoplasmic reticulum membrane, isolated at the earliest stages of development analysed. Its amount increases continuously with the rabbit's age to the adult value of about 70% of total sarcoplasmic reticulum protein. The low amount of 100000-dalton protein and lack of 4-nm surface particles in sarcoplasmic reticulum vesicles obtained from fetal and newborn rabbits are strictly correlated with the low activity of Ca2+-dependent ATPase and the ability to take up Ca2+. These activities rise in parallel with the age of the rabbits. On the other hand, Mg2+-dependent ATPase activity is very high at the early stages of development and declines continuously to a low value in sarcoplasmic reticulum from adult muscle. The sarcoplasmic reticulum membrane from fetal and newborn rabbits contains a higher amount of lipids as compared with the membrane present in the muscle of adult animals. The ratio of both phospholipid to protein and neutral lipid to protein decreases with the age of the rabbits. The composition of sarcoplasmic reticulum phospholipids also changes during development.     

HeLa cell plasma membranes. Changes in membrane protein composition during the cell cycle.

HeLa cells grown in suspension culture were synchronized by amethopterin block and thymidine reversal. In some cases an additional Colcemid block was used to obtain mitotic cells. From the various phases of the cell cycle, cells were harvested and the plasma membranes isolated. The membrane proteins were solubilized in sodium dodecyl sulphate and separated by gel electrophoresis in the presence of sodium dodecyl sarcosinate. About 35 protein bands, five of which were stained with periodic acid-Schiff reagent, appeared. Most of the bands were identical in all membrane preparations, but a few minor bands seemed to be associated with limited periods of the cell cycle. In particular, the cells in mitosis apparently contained plasma membrane proteins which did not occur in other phases. Amino acid analyses of the plasma membranes revealed no significant cell cycle-dependent changes in the amino acid composition.     

Alterations in cell membrane properties caused by perfluorinated compounds

The recent detection of perfluorinated compounds (PFCs) in wildlife from even remote locations has spurred interest in the environmental occurrence and effects of these chemicals. While the global distribution of PFCs is increasingly understood, there is still little information available on their effects on wildlife. The amphiphillic nature of PFCs suggests that their effects could be primarily on cell membranes. In this study we measured the effects of PFCs on membrane fluidity and mitochondrial membrane potential using flow cytometry and effects on membrane permeability using cell bioassay procedures (H4IIE, MCF-7, PLHC-1). Of the PFCs tested, only perfluorooctane sulfonic acid (PFOS) increased the permeability of cell membranes to the hydrophobic ligands used. Three PFCs were tested in the membrane fluidity assay: PFOS, perfluorohexane sulfonic acid (PFHS), and perfluorobutane sulfonic acid (PFBS). PFOS increased membrane fluidity in fish leukocytes in a dose-dependent fashion, while PFHS and PFBS had no effect in the concentration range tested. The lowest effective concentrations for the membrane fluidity effects of PFOS were 5-15 mg/l. Effects on mitochondrial membrane potential occurred in the same concentration range as effects on membrane fluidity. This suggests that PFOS effects membrane properties at concentrations below those associated with other adverse effects.     

Changes in membrane lipid composition of Mycoplasma capricolum affect the cell volume.

The cellular water volume of Mycoplasma capricolum was markedly increased by a decrease in the cholesterol-to-phospholipid molar ratio in the membrane. An increase in cell volume was also observed with the increase in the phospholipid cell membrane content obtained by the incorporation of exogenous phosphatidylcholine from the growth medium.     

Changes in the cell wall composition and structure of Streptococcus pyogenes during batch culture

Changes in S. pyogenes cells in the process of batch cultivation have been studied. The composition of S. pyogenes cell walls has been studied by amino acid analysis; besides, their resistance to enzymatic hydrolysis and the electric conductivity of cell-wall lysates have been determined at different phases of the growth of S. pyogenes. The molar amino acid composition, expressed in percent, is unrelated to the growth phase, while the content of amino acids in preparations changes in the process of growth and reaches its maximum in the middle and in the end of the logarithmic phase. At the same time the electric conductivity of cell-wall lysates reaches the minimum level at these growth stages. The authors suggest that additional electrically charged compositions are formed in the cell walls at the beginning of the logarithmic and stationary phases. A considerable increase in the initial rate of cell-wall lysis with muramidase has been found to occur at the end of the logarithmic phase. This difference in the initial rate in the initial rates of lysis of S. pyogenes cell walls at different growth phases decreases after previous treatment of the cell walls with streptolytin possessing proteolytic activity. Analysis of these data leads to a conclusion on the "loose" structure of the outer protein layer of the cell wall at the end of the logarithmic phase of the growth curve.     

Changes in E. coli cell envelope structure caused by uncouplers of active transport and colicin E1

It is of interest to inquire whether agents that uncouple or deenergize membranes cause concomitant structural changes. The agents considered here are the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and the bacteriocidal protein colicin E1, agents for which there is some precedent for believing that they interact with membranes. In intact E. coli ML 308-225 cells the inhibition of [¹⁴C]-proline active transport by FCCP increases with uncoupler concentration from ～ 20% at 2 μM to ～100% at 5 μM. The increase in the rotational relaxation time (ρ) of the cell-bound fluorescent probe N-phenyl-1-naphthylamine (PhNap)

1 Abbreviations: FCCP – carbonyl cyanide p-trifluoromethoxyphenylhydrazone; ANS – 8-anilino-1-naphthalenesulfonate; PhNap, N-phenyl-1-naphthylamine; EDTA – ethylenediaminetetraacetate.

and 8-anilino-1-naphthalene-sulfonate (ANS) under these conditions shows the same dependence on FCCP concentration. For cells treated with EDTA to remove part of the outer lipopolysaccharide layer, inhibition of proline transport and the increase in ρ value of ANS show the same dependence on FCCP concentration with saturation at 0.3 μM. EDTA treatment causes a large increase in the binding and rotational relaxation time of PhNap, the latter quantity approaching a value obtained with purified inner membrane. Similar effects are produced in untreated cells by 5 μM FCCP. It is concluded that (a) EDTA treatment removes a permeability barrier t o FCCP and PhNap in the outer membrane; (b) uncoupling by FCCP removes a similar permeability barrier to PhNap; (c) binding of amphiphilic ANS, assumed to be located in the outer membrane, is hardly changed by these treatments; (d) deenergization of the inner membrane by FCCP thus causes a structural change in the outer membrane as measured by the permeability change to hydrophobic PhNap and the increase in ρ values of the amphiphilic ANS; (e) The binding sites reached by PhNap within the permeability barrier at or near the inner membrane are changed by FCCP from their initial state. This is inferred from an increase in PhNap quantum yield extrapolated to infinite cell concentration, and from removal by FCCP of an apparent phase transition sensed by the PhNap rotational relaxation time. Thus, uncoupling and deenergization by FCCP appears to cause structural change both in the outer membrane and inside the permeability barrier of the outer membrane. Transmission of the colicin E1 response in the envelope of intact and EDTA-treated cells can also be monitored by an increase in ANS and PhNap fluorescence intensity, a smaller fractional increase in dye binding, and a large increase in probe rotational relaxation time. The fluorescence changes of ANS again imply structural effects in the outer membrane caused by colicin. The binding and fluorescence changes of PhNap caused by colicin E1 acting on intact cells again imply an effect of deenergization on the permeability barrier of the outer membrane. Fluorescence changes with PhNap in intact and EDTA-treated cells show that the dye binding sites are altered in the presence of colicin E1. It is also shown that the PhNap intensity change can be blocked by low concentrations of vitamin B12, which competes for the colicin E1 receptor. Some properties are presented of the probe chlorotetracycline, which has been proposed by others to be an indicator of magnesium. The probe appears to reside in an environment somewhat similar to that of ANS, but the colicin-induced changes in its fluorescence parameters appear to be small under our conditions.     

Changes in the composition of membrane phospholipids during the cell cycle of Escherichia coli

Phospholipid concentrations have been estimated throughout the successive cell cycle in synchronously growing culture of E. coli B/r. Total phospholipid phosphorus was shown to be doubled in the period of time between two cell divisions, whereas during the division itself it did not change. Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) exhibit a stepwise increase during the cell cycle. It should be noted that the phase of accumulation of these lipids could shift depending on the duration of the cell cycle. The fall in level of PE was followed by a short-term increase (5-10 min). At the same time the level of cardiolipin was observed to be significantly increased.     

腈菌唑导致的细胞膜穿孔对斜纹夜蛾SL细胞膜通透性的影响

研究了腈菌唑导致的细胞膜穿孔对离体培养斜纹夜蛾Spodoptera litura卵巢细胞(SL细胞)膜通透性的影响。结果表明: 腈菌唑可使不能穿过SL细胞膜的大分子物质荧光染料PI大量穿过细胞膜进入胞内,20和40 μg/mL腈菌唑处理后12 h,SL细胞荧光强度增加率分别为11.95%和25.80%;处理后24 h,SL细胞荧光强度增加率分别为27.77%和57.49%。腈菌唑也可明显提高SL细胞内大分子物质乳酸脱氢酶的漏出率,20和40 μg/mL腈菌唑处理SL细胞24 h后,胞内乳酸脱氢酶漏出率分别为30.66%和43.93%;处理后48 h,漏出率分别为41.22%和57.91%。腈菌唑可以明显提高SL细胞胞内钙离子含量,20和40 μg/mL腈菌唑处理SL细胞24和48 h,胞内钙荧光强度与对照差异显著。处理后24 h,腈菌唑对SL细胞的LC₅₀值为35.84 μg/mL,明显高于典型细胞毒剂鱼藤酮的活性。当质量比为1∶1和2∶1时,腈菌唑与鱼藤酮联用对SL细胞处理后24 h的LC₅₀值为43.92和26.09 μg/mL,共毒系数分别为137.60和188.49,增效作用显著,随着腈菌唑的含量增加,增效作用增加。腈菌唑对SL细胞具有良好的抑制作用,可以打通限制物质穿透的细胞膜屏障,提高农药活性成分的有效利用率。     

Changes in lipid composition of hepatocyte plasma membrane induced by overfeeding in duck

This experiment was carried out to examine the influence of overfeeding ducks with corn on the lipid composition of hepatocyte plasma membrane. Seventy-day-old male Mule ducks (Cairina moschata × Anas platyrhynchos) were overfed with corn for 12.5 days in order to induce fatty livers. The cholesterol and phospholipid contents were approximately 50% higher in hepatocyte plasma membranes from fatty livers compared to those of lean livers obtained from non-overfed ducks. However, the cholesterol/phospholipids molar ratio did not differ between both groups. Overfeeding induced a significant change in phospholipid composition of hepatocyte plasma membrane with a decrease in phosphatidylcholine proportion and conversely an increase in phosphatidylethanolamine. The fatty acid profile of phospholipids was also altered. In fatty hepatocyte plasma membrane, the overall proportion of polyunsaturated fatty acids (PUFA) was decreased and this was due to the decrease of some of, but not all, the PUFA. In addition, the proportions of oleic acid and n-9 series unsaturated fatty acids were higher in fatty than in lean liver membranes. This study provides evidence that overfeeding with a carbohydrate-rich corn-based diet induces a de novo hepatic lipogenesis in Mule duck which predominates over dietary lipid intake to change the lipid composition of the hepatocyte plasma membrane.     

Flexible and digestible wood caused by viral-induced alteration of cell wall composition

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Changes in glycerolipids and their fatty acid composition during maturation of tobacco seeds

Triacylglycerol, which was one of the minor lipid components in immature seeds of tobacco, accumulated dramatically between 7 and 27 days after flowering and, in mature seeds at 37 days, the fatty acid methyl esters of the triacylglycerols comprised 96.3% of those of the total lipids. Diacylglycerols and sterol ester also increased significantly during seed development. Phosphatidylcholine and phosphatidylethanolamine, which were major components in immature seeds, decreased constantly with increasing maturation as well as the quantities of phosphatidylinositol and phosphatidylglycerol. Monogalactosyldiacylglycerols, digalactosyldiacylglycerols and sulfoquinovosyldiacylglycerols also decreased and disappeared in mature seeds. In the triacylglycerols the percentages of palmitate, stearate and linolenate fell with increasing seed age, while that of linoleate increased up to 75.3% in mature seeds. A similar trend was observed in the fatty acid composition in the diacylglycerols and sterol ester. Generally, in the phospholipids the proportions of linoleate and linolenate decreased with concomitant increases of stearate and oleate.     

Sertoli cell membrane polypeptide composition is modulated by germ cells

The polypeptide composition of Sertoli cell enriched cultures (SCEC) and Sertoli cell only cultures (SCOC) obtained after germ cell removal was investigated. Cells were labelled with [35S]methionine and analyzed by one and/or two-dimensional gel electrophoresis. The one-dimensional electrophoretic analysis of SCEC and SCOC particulate fraction did not show any appreciable difference between the two profiles. The more detailed two-dimensional electrophoretic analysis showed the appearance in SCOC of three polypeptides undetectable in SCEC. The molecular weight of these molecules ranged between 44 and 48 kDa. The effect of FSH on the Sertoli cell membrane composition was also investigated. No qualitative differences were detected, although the hormone increased many molecular species including the polypeptides appearing in SCOC. On the basis of these results, the hypothesis that germ cells influence Sertoli cell metabolic parameters is discussed.     

Changes in the composition of anionic membrane phospholipids influence protein secretion and cell envelope biogenesis in Escherichia coli

The secretion of alkaline phosphatase (PhoA) and peculiarities of biogenesis of the cell envelope were studied in Escherichia coli strains HD30/pHD102 and HDL11 with controlled synthesis of the anionic phospholipids, phosphatidylglycerol and cardiolipin. Inactivation of the pgsA gene responsible for the synthesis of anionic phospholipids or changes in the regulation of its expression by an environmental factor caused changes in the metabolism and composition of membrane phospholipids, which resulted in a decrease in the secretion of alkaline phosphatase through the cytoplasmic membrane and an increase in PhoA secretion from the periplasm into the culture medium. An increase was observed in exopolysaccharide secretion, as well as a decrease in the contents of the outer membrane lipopolysaccharides and lipopolyproteins, which determine its barrier properties. The results obtained show that anionic phospholipids play a significant role in protein secretion and are probably involved in the interrelation between protein secretion and biogenesis of cell envelope components.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 179–184.Original Russian Text Copyright © 2005 by Anisimova, Badyakina, Vasileva, Nesmeyanova.     

Changes in the structure of neuromuscular junctions caused by variations in osmotic pressure

Reconstituted cartilage collagen fibrils with an oblique banding pattern or with two types of symmetrical patterns, and reconstituted rattail tendon fibrils with a third type of symmetrical pattern were examined by electron microscopy and found to consist of narrow subfibrils having native-type cross-striations. Analysis of the four types of patterns by a graphic method of specific band matching revealed the orientation and axial relation of individual subfibrils and their component molecules. In fibrils with an oblique pattern, subfibrils have the same orientation and a regular 100A axial displacement. Observations on staining characteristics, folded fibrils, and transverse sections of embedded fibrils suggest that the obliquely banded fibrils are ribbonlike or layered structures. In the three types of fibrils with a symmetrical pattern, adjacent subfibrils are oppositely oriented and aligned within a 119-A segment of the 670-A major period. Considered together, the observations suggest that interaction sites on the surface of subfibrils (and perhaps on the surface of native collagen fibrils) occur in various patterns that are manifested accouding to the nature of the environment during fibril formation, and that such patterns can be mapped on the surface of subfibrils by noting the arrangement of subfibrils in polymorphic forms.     

Kinetin induced changes in cell wall composition of tobacco callus

Culture of tobacco callus on high or low kinetin in light or darkness leads to changed tissue texture and associated changes in cell wall composition. In particular, friable callus (low kinetin, darkness) cell walls have a greater extensin content and an altered composition of arabinose and xylose containing hemicelluloses compared with cell walls of compact callus (high kinetin, darkness). The possible importance of these differences in determining callus friability is discussed.