Biophysical parameters of the Sec14 phospholipid exchange cycle - Effect of lipid packing in membranes.

Sec14, a yeast phosphatidylinositol/phosphatidylcholine transfer protein, functions at the trans-Golgi membranes. It lacks domains involved in protein-protein or protein-lipid interactions and consists solely of the Sec14 domain; hence, the mechanism underlying Sec14 function at proper sites remains unclear. In this study, we focused on the lipid packing of membranes and evaluated its association with in vitro Sec14 lipid transfer activity. Phospholipid transfer assays using pyrene-labelled phosphatidylcholine suggested that increased membrane curvature as well as the incorporation of phosphatidylethanolamine accelerated the lipid transfer. The quantity of membrane-bound Sec14 significantly increased in these membranes, indicating that "packing defects" of the membranes promote the membrane binding and phospholipid transfer of Sec14. Increased levels of phospholipid unsaturation promoted Sec14-mediated PC transfer, but had little effect on the membrane binding of the protein. Our results demonstrate the possibility that the location and function of Sec14 are regulated by the lipid packing states produced by a translocase activity at the trans-Golgi network.

Biophysical Parameters of the Sec14 Phospholipid Exchange Cycle.

Sec14, the major yeast phosphatidylcholine (PC)/phosphatidylinositol (PI) transfer protein (PITP), coordinates PC and PI metabolism to facilitate an appropriate and essential lipid signaling environment for membrane trafficking from trans-Golgi membranes. The Sec14 PI/PC exchange cycle is essential for its essential biological activity, but fundamental aspects of how this PITP executes its lipid transfer cycle remain unknown. To address some of these outstanding issues, we applied time-resolved small-angle neutron scattering for the determination of protein-mediated intervesicular movement of deuterated and hydrogenated phospholipids in vitro. Quantitative analysis by small-angle neutron scattering revealed that Sec14 PI- and PC-exchange activities were sensitive to both the lipid composition and curvature of membranes. Moreover, we report that these two parameters regulate lipid exchange activity via distinct mechanisms. Increased membrane curvature promoted both membrane binding and lipid exchange properties of Sec14, indicating that this PITP preferentially acts on the membrane site with a convexly curved face. This biophysical property likely constitutes part of a mechanism by which spatial specificity of Sec14 function is determined in cells. Finally, wild-type Sec14, but not a mixture of Sec14 proteins specifically deficient in either PC- or PI-binding activity, was able to effect a net transfer of PI or PC down opposing concentration gradients in vitro.

Kinetic Analysis of the Methyl-ß-cyclodextrin-Mediated Intervesicular Transfer of Pyrene-Labeled Phospholipids.

Methyl-ß-cyclodextrin (MßCD) can transfer phospholipids between vesicles, and its transfer ability has been utilized for the preparation of asymmetric vesicle and lipid incorporation into culture cells. Nevertheless, a detailed kinetic analysis of the MßCD-mediated phospholipid transfer has not yet been carried out. We performed real-time monitoring of intervesicular lipid transfer by means of the fluorescence of pyrene-labeled phospholipids. Intermolecular excimer formation of the pyrene-labeled lipids in a membrane strongly depends on the local concentration of the fluorophore and decreases when the pyrene-labeled lipids are transferred from donor (fluorophore-containing) vesicles to acceptor (fluorophore-free) vesicles. We monitored the fluorescence intensity of the pyrene monomer and excimer simultaneously and found that the excimer/monomer ratio decreased in the presence of MßCD, pointing to MßCD-mediated lipid transfer. The transfer rate depended on the MßCD concentration but not on the lipid concentration, suggesting that dissociation from the membrane via extraction by MßCD is the rate-limiting step of the lipid transfer. Calibration of the excimer/monomer ratio to the molar fraction of the pyrene-labeled lipids enabled us to evaluate the dissociation rate constant correctly. From the temperature dependence of the transfer, we obtained the thermodynamic activation parameters, which revealed that the extraction of phosphatidylcholine by MßCD from membranes is less enthalpically unfavorable than that of phosphatidylglycerol and phosphatidylinositol.

Reliability and validity of the self-report Quality of Life Questionnaire for Japanese School-aged Children with Asthma (JSCA-QOL v.3).

BACKGROUND: Asthma is a chronic disease prevalent in children which threatens their quality of life (QOL) through unexpected asthma attacks and/or the burden of daily self-management. As some conditions of chronic illness make it difficult for a child to accomplish normal developmental tasks, there may be fewer opportunities for the child to obtain a sense of achievement. This study investigated the reliability and validity of the Quality of Life Questionnaire for Japanese School-aged Children with Asthma Version 3 (JSCA-QOL v.3). This questionnaire includes 25 items with a 5-point Likert Scale format over five domains: "asthma attack triggers", "change in daily life", "family support", "satisfaction with daily life" and "restriction in participating in daily activities", and one summary scale. METHODS: In the present study, 2,425 children with asthma aged from 10 to 18 years were investigated in Japan. The internal consistency reliability of each domain was investigated with Cronbach's alpha reliability coefficient, and test-retest reliability with Spearman's correlations coefficient. Factorial validity by factor analysis using maximum-likelihood extraction with promax rotation was performed. Data analysis was performed using SPSS 12.0J. RESULTS: The final number of effective replies was 2,097 (the rate of effective data was 86.5%). "Asthma attack triggers", "change in daily life", "family support", "satisfaction with daily life" and "restriction in participating in daily activities" showed a high internal consistency (Cronbach's alpha = 0.66-0.86) as well as good test-retest reliability (Spearman's rho = 0.60, p < 0.01). The factorial validity was appropriate (KMO value = 0.90), because it was conceivable that the five factors extracted from factor analysis would be the same as in our hypothesis and support constructive validity. In addition, there was good correlation between the summary scale and the total QOL score (Spearman's rho = 0.58, p < 0.01). CONCLUSIONS: The present study showed that the JSCA-QOL v.3 is a reliable and valid measurement tool that can be used to appropriately assess QOL in school-aged children with asthma. As the JSCA-QOL v.3 can be easily completed in about 10 minutes, it can contribute as an efficient evaluation tool of the outcome of medical treatment through continual utilization in the outpatient clinic. The JSCA-QOL v.3 allows a health provider to help school-aged children with asthma to achieve their developmental tasks.