Effects of reduction of Rab activity on localisation of Comm and Robo expressed in S2 cells.

<p>A) Representative images of Robo-Venus and Comm-mCherry in transfected S2 cells (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064427#s2" target="_blank">Material and Methods</a>). The top panels show transfected cells with tagged version of either Robo or Comm. Robo is predominantly localized on membranes, but in some cells an additional vesicular component can be observed (indicated ‘membrane’ and ‘mem/ves’). Comm is always present in vesicles. The bottom panels show a co-transfection of Robo with Comm resulting in a complete exclusion of Robo from the plasma membrane. B) Quantification of the effect of reducing Rab function on the localisation of Robo in S2 cells in the presence or absence of Comm. Dominant negative constructs of Rab5, Rab7 or Rab11 have no effect on the Comm driven relocalisation of Robo. However, when Comm is not present, a reduction of Rab11 and Rab4 leads to a failure to accurately target Robo to the cell surface and Robo is found within intracellular vesicles (p<0.05).</p

Comm activity is dependent on PPCY/LPSY motifs but independent of ubiquitin attachment.

<p>Immunostainings of the <i>Drosophila</i> embryonic central nervous system to reveal the activity of the indicated UAS-Comm constructs when expressed pan-neuronally by elav-Gal4. Comm is the wild type allele, CommK>R is a construct where all intracellular lysines have been mutated to arginines to prevent ubiquitination and Comm2PY>AY contains mutations in both PPCY and LPSY motifs. A) CNS axons in stage 15 embryos visualised with the BP102 antibody. B) Longitudinal axons in stage 16 embryos within the CNS revealed with MAb 1D4. The right-most panels in A and B show co-staining (green) of the HA-epitope present on the Comm2PY>AY construct to confirm its expression by elavGal4.</p

Axonal guidance is not affected by expression of dominant negative regulators of endosomal trafficking.

<p>A) Comm is required for the formation of commissures. BP102 staining (red) reveals the normal pattern of axon pathways in the wild type <i>Drosophila</i> CNS (red). Commissural neurons (arrows) fail to form in the majority of segments in a <i>comm</i> mutant although the occasional commissure may form (arrowhead). Eg-Gal4 driving expression of CD8::GFP reveals a subset of commissural neurons (green) which fail to cross the midline in <i>comm</i> mutant embryos. B) Drosophila embryos were obtained from crosses of indicated dominant negative constructs with driver lines for expression in commissural (eg-Gal4), longitudinal (RN2-Gal4) neurons or with a pan-neuronal driver (elav-Gal4). The lines RN2- and eg-Gal4 also express CD8::GFP to enable identification of axons. Embryos were stained with anti-GFP (green) and counterstained (red) with either BP102 or 1D4 (in the case of RN2-Gal4). RN2-Gal4 neurons drive expression in longitudinally projecting eve-neurons <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064427#pone.0064427-Landgraf1" target="_blank">[25]</a>. Expression of the dominant negative constructs using these drivers does not disrupt axon outgrowth and guidance although some minor defects do occur (arrowhead). C) Expression of Comm in elav-neurons results in a <i>roundabout</i> phenocopy where longitudinal neurons cross the midline. This gain of activity of Comm is not affected by the co-expression of Rab5^DN suggesting Rab5 is not required for Comm activity. D) Quantification of the ability to form commissures in <i>comm</i> mutant embryos or embryos expressing dominant negative forms of Rab5, Rab7 or Shibire reveal that commissural neuron outgrowth is independent of Rab5, Rab7 and Shibire activity.</p

Localisation of Comm expression within commissural neurons with respect to intracellular compartment markers.

<p>A) Confocal images of Comm-mCherry and different intracellular compartment markers, labelled by GFP or YFP, expressed in eagle-neurons of stage 15 embryos. Constructs contain an UAS element and were driven by the egGal4 line, which drives expression in a specific subset of commissural neurons <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064427#pone.0064427-Higashijima1" target="_blank">[15]</a>. The cell bodies of a single EG cluster is shown in each panel. Comm colocalises with Rab7 and Shrub (arrowheads). B) Quantification of the vesicle pool that shows colocalisation between the red and green channels. (i) Red bars indicate the percentage of Comm vesicles that are positive for a specific marker out of the total pool of Comm vesicles. (ii) Green bars indicate the percentage of vesicles that express a specific marker which also overlap a Comm vesicle out of the total pool of marker vesicles. The percentages of colocalisation of firstly Comm with Rab7 (red bars) and secondly Shrub-GFP with Comm (green bars) are significantly different from each other compartments in their respective groups (at least P<.01, one-way ANOVA with a Tukey-Kramer post test).</p

Hierarchy levels, sum score, and worsening of disability are related to depressive symptoms in elderly men from three European countries

Objectives: The objectives were to investigate the predictive value of hierarchy levels and sum score of disability and change in disability on depressive symptoms. Method: Longitudinal data of 723 men age 70 and older from the Finland, Italy, and the Netherlands Elderly Study were collected in 1990 and 1995. Self-reported disability was based on three disability domains (instrumental activities, mobility, and basic activities) and depressive symptoms on the Zung questionnaire. Results: Severity levels of disability were positively associated with depressive symptoms. Men with no disability scored 5 to 17 points lower (p < .01) on depressive symptoms than did those with disability in all domains. Among men with mild disability, those who had worsening of disability status in the preceding 5 years scored 5 points higher (p = .004) on depressive symptoms than did men who improved. Discussion: Hierarchic severity levels, sum score of disability, and preceding changes in disability status are risk factors for depressive symptom

Characterization of the final step in the conversion of phytol into phytanic acid

Phytol is a branched-chain fatty alcohol that is a naturally occurring precursor of phytanic acid, a fatty acid involved in the pathogenesis of Refsum disease. The conversion of phytol into phytanic acid is generally believed to take place via three enzymatic steps that involve 1) oxidation to its aldehyde, 2) further oxidation to phytenic acid, and 3) reduction of the double bond at the 2,3 position, yielding phytanic acid. Our recent investigations of this mechanism have elucidated the enzymatic steps leading to phytenic acid production, but the final step of the pathway has not been investigated so far. In this study, we describe the characterization of phytenic acid reduction in rat liver. NADPH-dependent conversion of phytenic acid into phytanic acid was detected, although at a slow rate. However, it was shown that phytenic acid can be activated to its CoA ester and that reduction of phytenoyl-CoA is much more efficient than that of phytenic acid. Furthermore, in rat hepatocytes cultured in the presence of phytol, phytenoyl-CoA could be detected, showing that it is a bona fide intermediate of phytol degradation. Subcellular fractionation experiments revealed that phytenoyl-CoA reductase activity is present in peroxisomes and mitochondria. With these findings, we have accomplished the full elucidation of the mechanism by which phytol is converted into phytanic aci

Self-reported disability and its association with performance-based limitation in elderly men: A comparison of three European countries

OBJECTIVES: To compare self-reported disability and performance-based limitation and their association in elderly men from three European countries. DESIGN: Cross-sectional data from a cohort study collected around 1990. SETTING: Three cohorts from Finland, the Netherlands, and Italy. PARTICIPANTS: One thousand one hundred sixty-one men aged 70 and older. MEASUREMENTS: Disability and functional limitation were measured in a standardized way in three countries. Self-reported disability was estimated by questionnaire, assessing three domains of activities of daily living: instrumental activities of daily living, mobility, and activities of daily living (score 0-3). Functional limitation was measured by performance tests (score 0-16), with 0 indicated the healthiest score. RESULTS: Self-reported disability and performance-based limitation scores differed between countries. Mean self-reported disability score was worse in Italy (0.72) and the Netherlands (0.70) than in Finland (0.54). Italian men scored worst on the performance-based tests (mean 4.80 vs 4.04 for Finland and 3.74 for the Netherlands). Differences in self-reported disability remained after adjusting for performance scores: Dutch men reported more disabilities (odds ratio (OR) = 1.66, 95% confidence interval (CI) = 1.23-2.25) than men in Finland (reference group) and Italy (OR = 1.08, 95% CI = 0.77-1.53). Self-reported disability was positively associated with performance-based score (OR = 1.28, 95% CI = 1.21-1.35) and did not differ between countries. CONCLUSION: Cross-cultural variation was noted in self-reported disability adjusted for performance score. These differences may be due to sociocultural and physical environmental factors. Self-reported disability was consistently associated with performance-based limitation in Finland, the Netherlands, and Ital