Enhanced exposure of phosphatidylserine in human gastric carcinoma cells overexpressing the half-size ABC transporter BCRP (ABCG2).

Members of the ABC (ATP-binding cassette) transporter super-family are emerging to be involved in lipid transport. In the present study, we studied the organization of phospholipids in the plasma membrane of EPG85-257 human gastric carcinoma cells overexpressing BCRP (breast cancer resistance protein, ABCG-2), a half-size transporter belonging to the ABCG subfamily. A significantly increased plasma membrane association of the PS (phosphatidylserine)-binding probe FITC-Annexin V in comparison with control cells was observed. Treatment of BCRP -overexpressing cells with the inhibitor Tryprostatin A decreased FITC-Annexin V binding almost to the control level. This suggests an enhanced exposure of PS in BCRP -overexpressing cells, which is dependent on functional BCRP. A role of BCRP in the transverse distribution of lipids in the plasma membrane is supported by the increased outward transport of the lipid analogue C6- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PS in BCRP -overexpressing EPG85-257 cells and MCF-7 human breast cancer cells. As shown for BCRP -overexpressing EPG85-257 cells, enhanced outward redistribution of the lipid analogue is inhibited by Tryprostatin A as well as by reduction of BCRP expression on transfection with an anti- BCRP -ribozyme. We also observed an enhanced outward transport of C6- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phosphatidylcholine in BCRP -overexpressing EPG85-257 cells, suggesting that the influence of BCRP on transverse lipid organization is not highly specific

Extraction of nucleic acids from yeast cells and plant tissues using ethanol as medium for sample preservation and cell disruption

Here we report that dehydrated ethanol is an excellent medium for both in situ preservation of nucleic acids and cell disruption of plant and yeast cells. Cell disruption was strongly facilitated by prior dehydration of the ethanol using dehydrated zeolite. Following removal of ethanol, nucleic acids were extracted from the homogenate pellet using denaturing buffers. The method provided DNA and RNA of high yield and integrity. Whereas cell wall disruption was essential for extraction of DNA and large RNA molecules, smaller molecules such as tRNAs could be selectively extracted from undisrupted, ethanol-treated yeast cells. Our results demonstrate the utility of absolute ethanol for sample fixation, cell membrane and cell wall disruption, as well as preservation of nucleic acids during sample storage

Improved Metrological Traceability of Particle Size Values Measured with Line-Start Incremental Centrifugal Liquid Sedimentation

Line-start incremental centrifugal liquid sedimentation (disc-CLS) is a powerful method to determine particle size based on the principles of Stokes’ law. Because several of the input quantities of the Stokes equation cannot be easily determined for this case of a rotating disc, the disc-CLS approach relies on calibrating the sedimentation time scale with reference particles. To use these calibrant particles for establishing metrological traceability, they must fulfill the same requirements as those imposed on a certified reference material, i.e., their certified Stokes diameter and density value must come with a realistic measurement uncertainty and with a traceability statement. As is the case for several other techniques, the calibrants do not always come with uncertainties for the assigned modal diameter and effective particle density. The lack of such information and the absence of a traceability statement make it difficult for the end-user to estimate the uncertainty of the measurement results and to compare them with results obtained by others. We present the results of a collaborative study that aimed at demonstrating the traceability of particle size results obtained with disc-CLS. For this purpose, the particle size and effective particle density of polyvinyl chloride calibrants were measured using different validated methods, and measurement uncertainties were estimated according to the Guide to the Expression of Uncertainty in Measurement. The results indicate that the modal Stokes diameter and effective particle density that are assigned to the calibrants are accurate within 5% and 3.5%, respectively, and that they can be used to establish traceability of particle size results obtained with disc-CLS. This conclusion has a great impact on the traceability statement of certified particle size reference materials, for which the traceability is limited to the size and density values of the calibrant particles.JRC.F.6-Reference Material

Improved Metrological Traceability of Particle Size Values Measured with Line-Start Incremental Centrifugal Liquid Sedimentation

Line-start incremental centrifugal liquid sedimentation (disc-CLS) is a powerful method to determine particle size based on the principles of Stokes’ law. Because several of the input quantities of the Stokes equation cannot be easily determined for this case of a rotating disc, the disc-CLS approach relies on calibrating the sedimentation time scale with reference particles. To use these calibrant particles for establishing metrological traceability, they must fulfill the same requirements as those imposed on a certified reference material, i.e., their certified Stokes diameter and density value must come with a realistic measurement uncertainty and with a traceability statement. As is the case for several other techniques, the calibrants do not always come with uncertainties for the assigned modal diameter and effective particle density. The lack of such information and the absence of a traceability statement make it difficult for the end-user to estimate the uncertainty of the measurement results and to compare them with results obtained by others. We present the results of a collaborative study that aimed at demonstrating the traceability of particle size results obtained with disc-CLS. For this purpose, the particle size and effective particle density of polyvinyl chloride calibrants were measured using different validated methods, and measurement uncertainties were estimated according to the Guide to the Expression of Uncertainty in Measurement. The results indicate that the modal Stokes diameter and effective particle density that are assigned to the calibrants are accurate within 5% and 3.5%, respectively, and that they can be used to establish traceability of particle size results obtained with disc-CLS. This conclusion has a great impact on the traceability statement of certified particle size reference materials, for which the traceability is limited to the size and density values of the calibrant particles