Amino acid substitution equivalent to human chorea-acanthocytosis I2771R in yeast Vps13 protein affects its binding to phosphatidylinositol 3-phosphate

The rare human disorder chorea-acanthocytosis (ChAc) is caused by mutations in hVPS13A gene. The hVps13A protein interacts with actin and regulates the level of phosphatidylinositol 4-phosphate (PI4P) in the membranes of neuronal cells. Yeast Vps13 is involved in vacuolar protein transport and, like hVps13A, participates in PI4P metabolism. Vps13 proteins are conserved in eukaryotes, but their molecular function remains unknown. One of the mutations found in ChAc patients causes amino acids substitution I2771R which affects the localization of hVps13A in skeletal muscles. To dissect the mechanism of pathogenesis of I2771R, we created and analyzed a yeast strain carrying the equivalent mutation. Here we show that in yeast, substitution I2749R causes dysfunction of Vps13 protein in endocytosis and vacuolar transport, although the level of the protein is not affected, suggesting loss of function. We also show that Vps13, like hVps13A, influences actin cytoskeleton organization and binds actin in immunoprecipitation experiments. Vps13-I2749R binds actin, but does not function in the actin cytoskeleton organization. Moreover, we show that Vps13 binds phospholipids, especially phosphatidylinositol 3-phosphate (PI3P), via its SHR_BD and APT1 domains. Substitution I2749R attenuates this ability. Finally, the localization of Vps13-GFP is altered when cellular levels of PI3P are decreased indicating its trafficking within the endosomal membrane system. These results suggest that PI3P regulates the functioning of Vps13, both in protein trafficking and actin cytoskeleton organization. Attenuation of PI3P-binding ability in the mutant hVps13A protein may be one of the reasons for its mislocalization and disrupted function in cells of patients suffering from ChAc

pH tolerances and regulatory abilities of freshwater and euryhaline Aedine mosquito larvae

The pH regulatory abilities of two members of the mosquito tribe Aedini, known to have dramatically different saline tolerances, are investigated. The freshwater mosquito Aedes aegypti and the euryhaline Ochlerotatus taeniorhynchus tolerate very similar pH ranges. Both species complete larval development in waters ranging from pH·4 to pH·11, but naïve larvae always die in water of pH·3 or 12. Across the pH range 4–11, the hemolymph pH of O. taeniorhynchus is maintained constant while that of A. aegypti varies by 0.1 pH units. The salt composition of the water (3.5·g·l–1 sea salt, 3.5·g·l–1 NaCl, or nominally salt-free) has no effect on the range of pH tolerated by A. aegypti. In both speies, the effects of pH on larval growth and development are minor in comparison with the influence of species and sex. Acclimation of A. aegypti to pH·4 or 11 increases survival times in pH·3 or 12, respectively, and allows a small percentage of larvae to pupate successfully at these extreme pH values. Such acclimation does not compromise survival at the other pH extreme. Key words: mosquito larvae, pH regulation, pH acclimation, life history, Aedes aegypti, Ochlerotatus taeniorhynchu

Three-Dimensional Ultrasonography for the Evaluation of Atherosclerotic Stenoses of the Carotid Trunk

Three-dimensional ultrasonography is a significant novelty in neurosonology as it offers the possibility of an even more successful evaluation of atherosclerotic stenoses of the carotid trunk than previous ultrasonographic investigations. In 37 patients with signs of transitory ischemic attack and in 5 patients with reversible ischemic neurologic deficit we compared the findings of three-dimensional ultrasonography of carotid arteries on the neck with those of three-dimensional CT angiography. In 20 of these patients in which carotid thrombendarterectomy had been carried out, the findings of both diagnostic methods were also compared with the angiographic and operative findings. In 2 out of the total of 42 compared findings of three-dimensional ultrasonography, three-dimensional CT angiography and angiography of the carotid trunk, we assessed a difference in the evaluation of the degree of carotid stenosis while all other findings were in accordance as regards the evaluation of the degree of stenosis and plaque analysis. Of 20 operative findings, one showed insignificant deviations from the findings of both diagnostic methods. It is our opinion that three-dimensional ultrasonography is a reliable diagnostic method in evaluating atherosclerotic stenoses of carotid arteries. Every subtotal stenosis or internal carotid artery occlusion established by three-dimensional ultrasonography must be compared with three-dimensional CT angiography or classic angiography due to the objective possibility of false ultrasonographic interpretation and the significance of carotid thrombendarterectomy

Phosphatidylinositol-3-phosphate regulates response of cells to proteotoxic stress

Human Nedd4 ubiquitin ligase, or its variants, inhibit yeast cell growth by disturbing the actin cytoskeleton organization and dynamics, and lead to an increase in levels of ubiquitinated proteins. In a screen for multicopy suppressors which rescue growth of yeast cells producing Nedd4 ligase with an inactive WW4 domain (Nedd4w4), we identified a fragment of ATG2 gene encoding part of the Atg2 core autophagy protein. Expression of the Atg2-C1 fragment (aa 1074-1447) improved growth, actin cytoskeleton organization, but did not significantly change the levels of ubiquitinated proteins in these cells. The GFP-Atg2-C1 protein in Nedd4w4-producing cells primarily localized to a single defined structure adjacent to the vacuole, surrounded by an actin filament ring, containing Hsp42 and Hsp104 chaperones. This localization was not affected in several atg deletion mutants, suggesting that it might be distinct from the phagophore assembly site (PAS). However, deletion of ATG18 encoding a phosphatidylinositol-3-phosphate (PI3P)-binding protein affected the morphology of the GFP-Atg2-C1 structure while deletion of ATG14 encoding a subunit of PI3 kinase suppressed toxicity of Nedd4w4 independently of GFP-Atg2-C1. Further analysis of the Atg2-C1 revealed that it contains an APT1 domain of previously uncharacterized function. Most importantly, we showed that this domain is able to bind phosphatidylinositol phosphates, especially PI3P, which is abundant in the PAS and endosomes. Together our results suggest that human Nedd4 ubiquitinates proteins in yeast and causes proteotoxic stress and, with some Atg proteins, leads to formation of a perivacuolar structure, which may be involved in sequestration, aggregation or degradation of proteins

Multivariate investigation of parameters in the development and improvement of NiFe cells

In this article, we use a surface response approach to investigate the effect of bismuth sulphide as well as the compositions of PTFE in the overall columbic efficiency of a NiFe cell battery. Our results demonstrate that while bismuth sulphide favours the process of charge/discharge of a NiFe cell, the use of metallic bismuth only marginally influences coulombic efficiency. In addition we had found that the presence of the soluble bisulfide anion is not sufficient to increase coulombic efficiency in NiFe cells. © 2014 The Authors. Published by Elsevier B.V

Melatonin and its metabolites ameliorate UVR-induced mitochondrial oxidative stress in human MNT-1 melanoma cells

Melatonin (Mel) is the major biologically active molecule secreted by the pineal gland. Mel and its metabolites, 6-hydroxymelatonin (6(OH)Mel) and 5-methoxytryptamine (5-MT), possess a variety of functions, including the scavenging of free radicals and the induction of protective or reparative mechanisms in the cell. Their amphiphilic character allows them to cross cellular membranes and reach subcellular organelles, including the mitochondria. Herein, the action of Mel, 6(OH)Mel, and 5-MT in human MNT-1 melanoma cells against ultraviolet B (UVB) radiation was investigated. The dose of 50 mJ/cm2 caused a significant reduction of cell viability up to 48%, while investigated compounds counteracted this deleterious effect. UVB exposure increased catalase activity and led to a simultaneous Ca++ influx (16%), while tested compounds prevented these disturbances. Additional analysis focused on mitochondrial respiration performed in isolated mitochondria from the liver of BALB/cJ mice where Mel, 6(OH)Mel, and 5-MT significantly enhanced the oxidative phosphorylation at the dose of 10−6 M with lower effects seen at 10−9 or 10−4 M. In conclusion, Mel, 6(OH)Mel and 5-MT protect MNT-1 cells, which express melatonin receptors (MT1 and MT2) against UVB-induced oxidative stress and mitochondrial dysfunction, including the uncoupling of oxidative phosphorylation