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

LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum

<p>Abstract</p> <p>Background</p> <p>The phospholipids of the plant plasma membrane are synthesized in the endoplasmic reticulum (ER). The majority of these lipids reach the plasma membrane independently of the secretory vesicular pathway. Phospholipid delivery to the mitochondria and chloroplasts of plant cells also bypasses the secretory pathway and here it has been proposed that lysophospholipids are transported at contact sites between specific regions of the ER and the respective organelle, followed by lysophospholipid acylation in the target organelle. To test the hypothesis that a corresponding mechanism operates to transport phospholipids to the plasma membrane outside the secretory pathway, we investigated whether lysolipid acylation occurs also in the plant plasma membrane and whether this membrane, like the chloroplasts and mitochondria, is in close contact with the ER.</p> <p>Results</p> <p>The plant plasma membrane readily incorporated the acyl chain of acyl-CoA into phospholipids. Oleic acid was preferred over palmitic acid as substrate and acyl incorporation occurred predominantly into phosphatidylcholine (PC). Phospholipase A₂stimulated the reaction, as did exogenous lysoPC when administered in above critical micellar concentrations. AgNO₃was inhibitory. The lysophospholipid acylation reaction was higher in a membrane fraction that could be washed off the isolated plasma membranes after repeated freezing and thawing cycles in a medium with lowered pH. This fraction exhibited several ER-like characteristics. When plasma membranes isolated from transgenic <it>Arabidopsis </it>expressing green fluorescent protein in the ER lumen were observed by confocal microscopy, membranes of ER origin were associated with the isolated plasma membranes.</p> <p>Conclusion</p> <p>We conclude that a lysoPC acylation activity is associated with plant plasma membranes and cannot exclude a PC transacylase activity. It is highly plausible that the enzyme(s) resides in a fraction of the ER, closely associated with the plasma membrane, or in both. We suggest that this fraction might be the equivalent of the mitochondria associated membrane of ER origin that delivers phospholipids to the mitochondria, and to the recently isolated ER-derived membrane fraction that is in close contact with chloroplasts. The <it>in situ </it>function of the lysoPC acylation/PC transacylase activity is unknown, but involvement in lipid delivery from the ER to the plasma membrane is suggested.</p

Accurate measurement of endogenous adenosine in human blood.

Accurate determination of in vivo circulating concentrations of extracellular adenosine in blood samples is challenging due to the rapid formation and rapid clearance of adenosine in blood. A blood collection protocol was developed based on direct sampling of venous blood into, and instant mixing with, a STOP solution developed to conserve in vivo adenosine concentrations by completely preventing both its formation and clearance in collected blood. Stable isotope labeled AMP and adenosine spiked into blood ex vivo were used in combination with mass spectrometry to evaluate conservation of adenosine and prevention of its formation. A number of approved drugs, including the P2Y12 antagonist ticagrelor, have been described to increase extracellular adenosine. This may contribute to its clinical profile, highlighting the importance of accurate measurement of in vivo adenosine concentrations.A high sensitive ultra performance liquid chromatography-tandem- mass spectrometry (UPLC-tandem-MS) analytical method for plasma adenosine was developed and validated with a lower limit of quantification of 2 nmol/L. The method demonstrated plasma adenosine stability during sample processing and analytical method performance relevant to human blood samples. The final STOP solution proved able to conserve exogenous adenosine and to prevent adenosine formation from exogenous AMP added in vitro to human blood over 15 minutes. The mean endogenous adenosine concentration in plasma prepared from venous blood collected from 10 healthy volunteers was 13 ± 7 nmol/L. Finally, the method was used to demonstrate the previously described concentration-dependent ability of ticagrelor to conserve extracellular adenosine at clinically relevant exposures. In conclusion, we report an optimized sampling protocol and a validated analytical method for accurate measurement of in vivo circulating adenosine concentrations in human blood, suitable for use in clinical trials

LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum-3

<p><b>Copyright information:</b></p><p>Taken from "LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum"</p><p>http://www.biomedcentral.com/1471-2229/7/64</p><p>BMC Plant Biology 2007;7():64-64.</p><p>Published online 28 Nov 2007</p><p>PMCID:PMC2241621.</p><p></p>ained 25 μg plasma membrane protein. , Distribution of radiolabel between 18:1-CoA (solid squares), free fatty acids (open squares) and phospholipids (open circles). , Incorporation of radiolabel into PC (filled symbols) and PE (open symbols). Either [C]18:1-CoA (solid line) or [C]16:0-CoA (dashed line) was used as substrate. The data represent mean values ± the range of duplicates from a representative experiment

LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum-4

<p><b>Copyright information:</b></p><p>Taken from "LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum"</p><p>http://www.biomedcentral.com/1471-2229/7/64</p><p>BMC Plant Biology 2007;7():64-64.</p><p>Published online 28 Nov 2007</p><p>PMCID:PMC2241621.</p><p></p>centration of added lysophospholipid was 70 μM (lysoPC and lysoPE were predominantly acylated with 16:0 or 18:0, lysoPA was acylated with 18:1). Radiolabel recovery is presented for PC (solid bars), PE (cross-hatched bars) and PA (open bars). Otherwise as in Figure 4

LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum-10

<p><b>Copyright information:</b></p><p>Taken from "LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum"</p><p>http://www.biomedcentral.com/1471-2229/7/64</p><p>BMC Plant Biology 2007;7():64-64.</p><p>Published online 28 Nov 2007</p><p>PMCID:PMC2241621.</p><p></p>eous polymer two phase counter current distribution of pea seedling microsome membranes (1–10; cf. Fig. 1), pea plasma membrane (PM) and pea microsome membranes (MS). The arrows mark the positions of molecular weight markers