9 research outputs found
The Primary Folding Defect and Rescue of ΔF508 CFTR Emerge during Translation of the Mutant Domain
In the vast majority of cystic fibrosis (CF) patients, deletion of residue F508 from CFTR is the cause of disease. F508 resides in the first nucleotide binding domain (NBD1) and its absence leads to CFTR misfolding and degradation. We show here that the primary folding defect arises during synthesis, as soon as NBD1 is translated. Introduction of either the I539T or G550E suppressor mutation in NBD1 partially rescues ΔF508 CFTR to the cell surface, but only I539T repaired ΔF508 NBD1. We demonstrated rescue of folding and stability of NBD1 from full-length ΔF508 CFTR expressed in cells to isolated purified domain. The co-translational rescue of ΔF508 NBD1 misfolding in CFTR by I539T advocates this domain as the most important drug target for cystic fibrosis
Psychometric properties of the Greek version of Jacelon Attributed Dignity Scale
Abstract
BACKGROUND:
Maintaining dignity is important for successful aging, but there is lack of validated research instruments in the nursing literature to investigate dignity as perceived by the old people.
OBJECTIVE:
This is a methodological study aiming to investigate the psychometric properties of the Greek version of Jacelon Attributed Dignity Scale as translated in the Greek language.
RESEARCH DESIGN:
A methodological approach consisting of translation, adaptation, and cross-cultural validation. A sample of 188 Greek-speaking old Cypriot persons drawn from the Hospital outpatient departments was asked to complete the Greek versions of Jacelon Attributed Dignity Scale and the Instrumental Activities of Daily Living. Data analyses included internal consistency reliability (Cronbach's alpha coefficient), item analysis, and exploratory factor analysis using principal component method with orthogonal varimax rotation.
ETHICAL CONSIDERATIONS:
The study protocol was approved by the National Bioethics committee according to the national legislation. Permission to use the research instrument was granted from the author. Information about the aim and the benefits of the study was included in the information letter.
FINDINGS:
Cronbach's alpha for Greek version of Jacelon Attributed Dignity Scale was 0.90. Four factors emerged explaining 65.28% of the total variance, and item to total correlation values ranged from 0.25 to 0.74 indicating high internal consistency and homogeneity. Mean item score in Instrumental Activities of Daily Living was 5.6 (standard deviation = 1.7) for men and 6.7 (standard deviation = 1.7) for women, and the correlations between demographics, Instrumental Activities of Daily Living, and the four factors of the Greek version of Jacelon Attributed Dignity Scale were low; also in multiple linear regression, the values of R2 are presented low.
DISCUSSION:
Demographic characteristics and degree of functionality seem to be associated with some of the dimensions of dignity but with low correlations; therefore, they cannot predict attributed dignity.
CONCLUSION:
The Greek version of Jacelon Attributed Dignity Scale is a valid and reliable tool to measure attributed dignity in Greek-speaking older adults, but further testing of the psychometric properties and other potential factors that may affect the attributed dignity is needed
Suppressing the ΔF508 phenotype <i>in vivo</i>.
<p>HeLa cells transiently expressing CFTR with the indicated mutations were pulse-labeled with <sup>35</sup>S-methionine and cysteine for 15 minutes and chased for the indicated times. CFTR molecules were immunoprecipitated from radiolabeled lysate using polyclonal antibody directed against the R-domain (G449). Samples were analyzed using 7.5% SDS-PAGE. The arrowhead indicates the ER oligomannose form (B-band) and the double arrowhead indicates complex glycosylated CFTR (C-band).</p
Co-translational misfolding and rescue of NBD1.
<p>(A) After 5 min pre-warming the translation mix, we added <sup>35</sup>S-methionine and followed CFTR synthesis in the SP-cell system for 10–60 min. Analysis using 10% SDS-PAGE directly visualized CFTR nascent chain elongation with time. Full-length ΔF508 CFTR (<) first appeared after 30 min of translation, “⧫” indicates persistent unfinished nascent chains. (B) Wild-type and ΔF508 nascent chains were translated <i>in vitro</i> and harvested after 20, 30, or 60 min of synthesis. All nascent chains were subjected to increasing proteinase K concentrations and proteolytic fragments were separated by 12% SDS-PAGE. In the 5 µg/ml proteinase K treatment shown here, the NBD1-related 25 kDa fragment is marked by an arrowhead. The bracket indicates increased protease resistance of CFTR domains as a result of nascent chain elongation. (C) Similar experimental conditions as in B but with the I539T mutation in wild-type and ΔF508 CFTR background. Nascent chains were harvested after 30 min of synthesis and protease resistant fragments were quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015458#pone-0015458-g001" target="_blank">Figure 1B</a>. Arrowhead indicates the NBD1-related 25 kDa fragment.</p
Rescue of NBD1 conformation by the I539T suppressor mutation.
<p>(A) Wild-type and ΔF508 NBD1 (top panel) mRNAs containing the G550E (middle panel) or I539T (bottom panel) mutation were <i>in vitro</i> translated in the presence of <sup>35</sup>S-labeled methionine and cysteine and analyzed by 15% SDS-PAGE after proteinase K treatment. Asterisk indicates the 27 kDa fragment, arrowhead indicates the 25 kDa fragment. (B) Longer exposure of the 100 µg/ml proteinase K digest of <i>in vitro</i> translated NBD1, from same experiment as shown in B, showing the rescue of the 17 kDa band by the I539T but not by the G550E mutation. Gel lanes are aligned on the 25 kDa bands. (C) CFTR molecules containing the indicated mutations were <i>in vitro</i> translated, analyzed using 12% SDS-PAGE and lanes were quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015458#pone-0015458-g001" target="_blank">Figure 1B</a>. The arrowhead indicates the 25 kDa fragment, which has slightly decreased mobility when the I539T mutation is present.</p
Stability of ΔF508 NBD1 is restored by introducing I539T.
<p>(A) Thermal denaturation of NBD1 variants, as indicated in the Figure. Bars represent average (n = 3) melting temperatures (T<sub>M</sub>), error-bars are SD. (B) Crystal structure of mouse NBD1 (1R0W). F508, T539 and G550 are shown in blue, and ATP in yellow. (C) Aligning CFTR sequences of several species revealed that an isoleucine or threonine on position 539 is species dependent. CFTR sequences are from <i>Oryctolagus cuniculus</i> (rabbit), <i>Xenopus laevis</i> (frog), <i>Rattus norvergicus</i> (rat), <i>Mus musculus</i> (mouse), <i>Homo sapiens</i> (human), <i>Pan troglodytes</i> (chimpanzee), <i>Equus caballus</i> (horse), <i>Canis lupus familiaris</i> (dog), <i>Mustela putorius furo</i> (ferret), <i>Sus scrofa</i> (pig). (D) CHO cells transiently expressing NBD1 with and without indicated mutations were pulse-labeled for 5 minutes and chased for 4 hours as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015458#pone-0015458-g002" target="_blank">Figure 2E</a>. The percentage of labeled NBD1 present after 4 hours of chase was quantified (n = 3, data represented ± SEM).</p
Minimal and local misfolding of ΔF508 CFTR.
<p>(A) Both CFTR and ΔF508 CFTR were translated <i>in vitro</i> in the presence of <sup>35</sup>S-methionine and cysteine and semi-permeabilized HT1080 cells for 60 min. Cells containing radiolabeled CFTR proteins were washed, lysed in Triton X-100, and prepared for limited proteolysis using increasing concentrations of proteinase K. The proteolytic digests were analyzed by 12% SDS-PAGE. The conformational difference between wild-type CFTR and ΔF508 CFTR is indicated by an arrowhead. (B) Relative intensities of all protease resistant fragments from a total 5 µg/ml Proteinase K digest, as in Figure 1A, were determined by total lane quantitation (Quantity One software Biorad). The y-axis represents electrophoretic mobility in 12% SDS-PA gel and the x-axis the relative intensity of the protease resistant fragments. The horizontal lines indicate the structural differences as described in A. The horizontal line indicated with an asterisk represents yet unidentified changes in the proteolytic pattern as a result of the ΔF508 mutation. The bracket represents small proteolytic fractions detected in both mutants. (C) Wild-type and ΔF508 CFTR were synthesized as in a, were subjected to 5 µg/ml proteinase K and NBD1-originated fragments were immunoprecipitated with polyclonal antibodies directed against NBD1 (Mr Pink) or against the R-region (G449). Arrowhead marks the NBD1-related fragment.</p
The effect of ΔF508 mutation on NBD1 alone.
<p>(A) Wild-type and ΔF508 NBD1 were <i>in vitro</i> translated for 30 min, treated with indicated proteinase K concentrations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015458#pone-0015458-g001" target="_blank">Figure 1</a>, and analyzed using 15% SDS-PAGE. The full length NBD1 domain is indicated by “#”, the asterisk (*) indicates the 27 kDa fragment, arrowhead (◂) indicates the 25 kDa fragment. (B) Similar experimental conditions as described in A, but using TPCK-trypsin as protease. A bracket (]) marks the triplet of protease resistant fragments and the dot (•) marks the 17 kDa fragment. (C) Wild-type and ΔF508 NBD1 were synthesized as in A, treated with 100 µg/ml trypsin, and fragments were immunoprecipitated with antibody 7D12 against NBD1. Fragments are labeled similar as in B. (D) Wild-type NBD1 was synthesized as in A, treated with 25 µg/ml proteinase K, and fragments were immunoprecipitated with the 7D12, 3G11 and Mr Pink antibody, recognizing specific epitopes within NBD1. Fragments are labeled similar as in A. (E) CHO cells expressing wild-type or ΔF508 NBD1 were pulse-labeled with <sup>35</sup>S-methionine and cysteine for 5 min and chased for indicated times. NBD1 was immunoprecipitated using polyclonal antibody Mr Pink and analyzed using 15% SDS-PAGE. NBD1 indicated by “#”. (F) Purified human wild-type and ΔF508 NBD1, indicated by “#”, were incubated with 2 µg/ml Proteinase K for 0, 2, 5, 10 and 30 minutes at room temperature. Proteolytic digests were separated using 15% SDS-PAGE and visualized by silver staining. Asterisk (*) and arrowhead (◂) indicate 27 and 25 kDa fragments resp., and are similar as in A. The open arrowhead (<) indicates a protease resistant background band in the purified ΔF508 NBD1.</p