Inside-out plant plasma membrane vesicles of high purity obtained by aqueous two-phase partitioning

AbstractHighly purified plasma membranes obtained from leaves of sugar beet (Beta vulgaris L.) by aqueous two-phase partitioning were separated into two fractions by further phase partition steps. The vesicles partitioning to the interface showed an ATP-dependent H+-uptake (measured using the pH probe acridine orange) and a negligible K+,Mg2+-ATPase latency, while the vesicles partitioning in the upper phase showed only slow H+-uptake and a high ATPase latency on addition of Triton X-100. Based on these results the material at the interface is estimated to contain ∼90% sealed, inside-out vesicles, and the material in the upper phase ∼90% sealed, right-side-out vesicle

Trichoderma viride cellulase induces resistance to the antibiotic pore-forming peptide alamethicin associated with changes in the plasma membrane lipid composition of tobacco BY-2 cells

<p>Abstract</p> <p>Background</p> <p>Alamethicin is a membrane-active peptide isolated from the beneficial root-colonising fungus <it>Trichoderma viride</it>. This peptide can insert into membranes to form voltage-dependent pores. We have previously shown that alamethicin efficiently permeabilises the plasma membrane, mitochondria and plastids of cultured plant cells. In the present investigation, tobacco cells (<it>Nicotiana tabacum </it>L. cv Bright Yellow-2) were pre-treated with elicitors of defence responses to study whether this would affect permeabilisation.</p> <p>Results</p> <p>Oxygen consumption experiments showed that added cellulase, already upon a limited cell wall digestion, induced a cellular resistance to alamethicin permeabilisation. This effect could not be elicited by xylanase or bacterial elicitors such as flg22 or elf18. The induction of alamethicin resistance was independent of novel protein synthesis. Also, the permeabilisation was unaffected by the membrane-depolarising agent FCCP. As judged by lipid analyses, isolated plasma membranes from cellulase-pretreated tobacco cells contained less negatively charged phospholipids (PS and PI), yet higher ratios of membrane lipid fatty acid to sterol and to protein, as compared to control membranes.</p> <p>Conclusion</p> <p>We suggest that altered membrane lipid composition as induced by cellulase activity may render the cells resistant to alamethicin. This induced resistance could reflect a natural process where the plant cells alter their sensitivity to membrane pore-forming agents secreted by <it>Trichoderma spp</it>. to attack other microorganisms, and thus adding to the beneficial effect that <it>Trichoderma </it>has for plant root growth. Furthermore, our data extends previous reports on artificial membranes on the importance of lipid packing and charge for alamethicin permeabilisation to <it>in vivo </it>conditions.</p

The association of actin and tubulin with plasma membranes: characterization using inside-out vesicles formed by Brij 58

Most processes of eukaryotic cells depend on the cortical cytoskeleton (CS), a protein filament structure associated to the plasma membrane (PM). With animal cells, much information has been collected on the mechanisms behind CS-PM interactions, but for plant cells the CS-PM links are poorly characterized. To allow investigations on these links, isolated PM from cauliflower were here treated with Brij 58, a detergent that causes the PM vesicles to turn inside-out (cytoplasmic side-out), thereby exposing the CS components. When actin and tubulin co-pelleted with inside-out PM were separated using sucrose gradient centrifugation, actin and tubulin were recovered with PM-marker activities, supporting intact links between these CS proteins and the Brij-treated PM. Inside-out PM was also treated with different media to learn more about the CS-PM interaction. Extensive dialysis against a low ionic strength medium released actin but not tubulin from these PM, while dialysis against 0.7 M NaCl had no effect. Neither 50 mM DTT, 10 mM CaCl2 nor 2 M NaCl had any effect on the release of either actin or tubulin from PM, but actin was completely released with 6 M urea or 0.6 M KI. Tubulin was also released by urea but not by KI. Incubation of PM in sodium carbonate at increasing pH led to a total release of actin at pH 10, of α-tubulin at pH 11 and of β-tubulin at pH 11.4. In many respects, these characteristics agree with reported findings using e.g., fluorescence microscopy with protoplast ghosts, suggesting that inside-out vesicles obtained with Brij 58 can be used in investigations aimed at understanding the role of the cortical CS in regulating PM-bound components

Cytoskeleton components of inside-out and right-side-out plasma membrane vesicles from plants

Isolated plasma membrane vesicles purified by aqueous polymer two-phase partitioning were used as a model system for studies on the membrane-associated (cortical) cytoskeleton in plants. Actin, as identified by immunoblotting, was found to be specifically attached to plasma membrane vesicles from cauliflower (Brassica oleracea L.). The actin was not washed off as the vesicles were turned inside-out, indicative of a fairly strong attachment. Triton X-100 extraction of plasma membrane vesicles resulted in an insoluble and hence pelletable fraction where actin could be found together with several other proteins. Our results show that the cortical cytoskeleton is to some extent co-purified with the plasma membrane, and we believe that well defined, inside-out and right-side-out plasma membrane vesicles can be used to study the structure and dynamics of the plant cortical cytoskeleton

Light-induced absorption changes in etiolated Coleoptiles

Blue (or green) light induced reversible and irreversible absorption changes in etiolated wheat (Triticum aestivum) coleoptiles. Immediately after a 10 s pulse of blue light, there was an absorbance increase at 440 nm and a decrease at 420 nm. The absorbance at 440 nm returned to the original level in a biphasic way with 1st-order rate constants of 0.05 s-1 and 0.006 s-1 at 25.degree. C. The change at 440 nm was partly, but not completely, inhibited by iodide. In the 500-600 nm region, complex changes occurred, indicating the participation of at least 2 different cytochromes, one of which was oxidized during a 10 s light pulse and the other oxidized more slowly during the following dark period

Localization of donor and acceptor sites of NADH dehydrogenase activities using inside-out and right-side-out plasma membrane vesicles from plants

Inside-out and right-side-out plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves, prepared by aqueous two-phase partitioning, were used to localize donor and acceptor sites and to determine substrate affinities for plasma membrane-bound NADH dehydrogenase activities. NADH-ferricyanide and NADH-cytochrome c reductase activities were approx. 30% latent with inside-out vesicles and about 80% latent with right-side-out vesicles, indicating that both donor and acceptor sites for these activities are located on the cytoplasmic surface of the plasma membrane, and that a possible transplasma membrane electron transport would constitute only a minor proportion of the total activity

Arlanda Airport Safety Nets & Digital Ground Vehicle Control - Förstudie, Slutrapport

Syftet med förstudien är att utreda förutsättningarna för att genomföra ett valideringsprojekt som ska identifiera var säkerhetskulturen, operativa koncept och system behöver förbättras för att bättre förebygga och minska antalet intrång på ban- och taxibanor på Arlanda flygplats och därmed öka säkerheten. Arbetet med förstudien är utfört av LFV, SWEDAVIA, ELTEL och VIKTORIA Swedish ICT. I denna förstudie till ett möjligt projekt vill vi fokusera på fordon och hur vi med ett eller flera stödsystem kan hjälpa våra fordonsförare att göra rätt d.v.s. minska antalet RWI (runway incursion) som kan leda till oönskade incidenter och olyckor. Vi vill även utöka forskningsområdet till att även omfatta taxiway incursion. På en flygplats med Arlandas komplexitet ställer det extra höga krav på fordonsförare att hålla reda på var de befinner sig för att inte av misstag köra ut på ett område utan klarering från tornet. Arbetsmiljön i fordonet bör vara enkel och tydlig för att underlätta för föraren. Stödsystemet ska inte ta onödig uppmärksamhet från föraren. Enkelhet gäller även för manövrering av stödsystemet i tornet. Förstudien syftar till att undersöka möjligheten att integrera kommunikationen, t.ex. reglering av trafikljus i befintlig teknik. Avsikten är att undvika behov av fler skärmar i tornet.Arlanda Airport Safety Nets and Runway Incursions Förstudie av ökad säkerhet på Arlanda flygplats gällande banintrån