Mitotic slippage in non-cancer cells induced by a microtubule disruptor, disorazole C1

<p>Abstract</p> <p>Background</p> <p>Disorazoles are polyene macrodiolides isolated from a myxobacterium fermentation broth. Disorazole C₁was newly synthesized and found to depolymerize microtubules and cause mitotic arrest. Here we examined the cellular responses to disorazole C₁in both non-cancer and cancer cells and compared our results to vinblastine and taxol.</p> <p>Results</p> <p>In non-cancer cells, disorazole C₁induced a prolonged mitotic arrest, followed by mitotic slippage, as confirmed by live cell imaging and cell cycle analysis. This mitotic slippage was associated with cyclin B degradation, but did not require p53. Four assays for apoptosis, including western blotting for poly(ADP-ribose) polymerase cleavage, microscopic analyses for cytochrome C release and annexin V staining, and gel electrophoresis examination for DNA laddering, were conducted and demonstrated little induction of apoptosis in non-cancer cells treated with disorazole C₁. On the contrary, we observed an activated apoptotic pathway in cancer cells, suggesting that normal and malignant cells respond differently to disorazole C₁.</p> <p>Conclusion</p> <p>Our studies demonstrate that non-cancer cells undergo mitotic slippage in a cyclin B-dependent and p53-independent manner after prolonged mitotic arrest caused by disorazole C₁. In contrast, cancer cells induce the apoptotic pathway after disorazole C₁treatment, indicating a possibly significant therapeutic window for this compound.</p

Membrane traffic and turnover in TRP-ML1–deficient cells: a revised model for mucolipidosis type IV pathogenesis

The lysosomal storage disorder mucolipidosis type IV (MLIV) is caused by mutations in the transient receptor potential–mucolipin-1 (TRP-ML1) ion channel. The “biogenesis” model for MLIV pathogenesis suggests that TRP-ML1 modulates postendocytic delivery to lysosomes by regulating interactions between late endosomes and lysosomes. This model is based on observed lipid trafficking delays in MLIV patient fibroblasts. Because membrane traffic aberrations may be secondary to lipid buildup in chronically TRP-ML1–deficient cells, we depleted TRP-ML1 in HeLa cells using small interfering RNA and examined the effects on cell morphology and postendocytic traffic. TRP-ML1 knockdown induced gradual accumulation of membranous inclusions and, thus, represents a good model in which to examine the direct effects of acute TRP-ML1 deficiency on membrane traffic. Ratiometric imaging revealed decreased lysosomal pH in TRP-ML1–deficient cells, suggesting a disruption in lysosomal function. Nevertheless, we found no effect of TRP-ML1 knockdown on the kinetics of protein or lipid delivery to lysosomes. In contrast, by comparing degradation kinetics of low density lipoprotein constituents, we confirmed a selective defect in cholesterol but not apolipoprotein B hydrolysis in MLIV fibroblasts. We hypothesize that the effects of TRP-ML1 loss on hydrolytic activity have a cumulative effect on lysosome function, resulting in a lag between TRP-ML1 loss and full manifestation of MLIV

PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. © 2013 Szalinski et al

VAMP7 modulates ciliary biogenesis in kidney cells

Epithelial cells elaborate specialized domains that have distinct protein and lipid compositions, including the apical and basolateral surfaces and primary cilia. Maintaining the identity of these domains is required for proper cell function, and requires the efficient and selective SNARE-mediated fusion of vesicles containing newly synthesized and recycling proteins with the proper target membrane. Multiple pathways exist to deliver newly synthesized proteins to the apical surface of kidney cells, and the post-Golgi SNAREs, or VAMPs, involved in these distinct pathways have not been identified. VAMP7 has been implicated in apical protein delivery in other cell types, and we hypothesized that this SNARE would have differential effects on the trafficking of apical proteins known to take distinct routes to the apical surface in kidney cells. VAMP7 expressed in polarized Madin Darby canine kidney cells colocalized primarily with LAMP2-positive compartments, and siRNA-mediated knockdown modulated lysosome size, consistent with the known function of VAMP7 in lysosomal delivery. Surprisingly, VAMP7 knockdown had no effect on apical delivery of numerous cargoes tested, but did decrease the length and frequency of primary cilia. Additionally, VAMP7 knockdown disrupted cystogenesis in cells grown in a three-dimensional basement membrane matrix. The effects of VAMP7 depletion on ciliogenesis and cystogenesis are not directly linked to the disruption of lysosomal function, as cilia lengths and cyst morphology were unaffected in an MDCK lysosomal storage disorder model. Together, our data suggest that VAMP7 plays an essential role in ciliogenesis and lumen formation. To our knowledge, this is the first study implicating an R-SNARE in ciliogenesis and cystogenesis. © 2014 Szalinski et al

Apport de la chirurgie dans la prise en charge du kyste du tractus thyréoglosse. A propos de 56 cas.

Les kystes du tractus thyréoglosse sont une pathologie fréquente en chirurgie infantile. Leur prise en charge dans les services spécialisés semble bien codifiée. Cependant il n'est pas rare de trouver encore des enfants multi opérés pour des tuméfactions cervicales mal étiquetées. Il nous a donc semblé intéressant d'étudier une série de 56 enfants opérés à l'Hôpital d'Enfants de Rabat dans le service de Chirurgie A , afin de faire une mise au point sur la prise en charge de ces enfants, ainsi que d'évaluer les risques de récidive. Cette série regroupant l’analyse des dossiers médicaux ainsi que l’étude des prélèvements anatomo-pathologiques, associés à une revue de la littérature, permet de clarifier cette prise en charge. Le diagnostic des kystes du tractus thyréoglosse est clinique. Le seul examen complémentaire nécessaire est l’échographie cervicale permettant d’éliminer le diagnostic de thyroïde ectopique. Le traitement est chirurgicale afin d’éviter les risques de surinfection. L’intervention doit se faire à distance d’un épisode infectieux selon la technique décrite par Sistrunk avec exérèse du corps de l’os hyoïde et résection d’un cône musculaire de base de langue. Les récidives malgré une technique bien conduite sont toujours possibles. Il semblerait que les infections préopératoires des kystes des tractus thyréoglosse soit un facteur de récidives

Microplastics in a Traditional Turkish Dairy Product: Ayran

Ingestion of microplastic particles (MP) through food has been associated with a multitude of health problems in humans. Although ayran is a traditional and nutritious Turkish beverage, the impact of microplastic pollution is unknown. This study examined the incidence of microplastic pollution on ayran by collecting samples throughout the production processes and the ingredients used to make ayran, including water, salt, cream, starting culture, cups, and lastly, the ayran. Optical and scanning electron microscope was applied for MP visualisation and measurement, and Fourier-transform infrared spectroscopy (FTIR) for polymer identification. Microplastics were detected in all examined filters except for the starter culture samples. The samples with the highest MP number were salty water (43 MP number/100 mL), salt (33 MP number/100 g), and milk samples taken from homogenization and pasteurization phases (26 MP number/100 mL). Additionally, 18 MP number/100 mL contamination was detected in the last product ayran. MP with a size range of 1–150 µm prevailed (37.38%). Ethylene propylene was the most frequently identified polymer in samples (39.30%). The findings of this study can help provide an overview of microplastic contamination in dairy production facilities and the potential human health risks associated with this microplastic exposure