ABSTRACT
To better understand the relationship between the subcellular compartmentalization of endothelial nitric oxide synthase (eNOS) and its function in endothelial cells, we addressed the roles of the microtubule network and of its dynamics in organizing Golgi-bound eNOS. We found that part of Golgi-bound eNOS localizes to the trans-Golgi network and/or to trans-Golgi network-derived vesicles and membrane tubules that are organized preferentially by stable microtubules. Also, while most of cellular eNOS was recovered in a detergent-resistant microtubule-enriched subcellular fraction, its recovery was impaired after total microtubule disassembly, but not after selective disassembly of dynamic microtubules or after microtubule stabilization. Basal eNOS phosphorylation on Ser(1177) further required the association of the trans-Golgi network to stable microtubules and was enhanced by microtubule stabilization. We finally show that the involvement of stable microtubules in basal eNOS phosphorylation involved alpha-tubulin acetylation. Microtubule-dependent organization of subcellular eNOS and control over its phosphorylation would thus be essential for endothelial cells to maintain their basal eNOS function.
Subject(s)
Endothelial Cells/metabolism , Microtubules/metabolism , Nitric Oxide Synthase Type III/metabolism , Phosphoserine/metabolism , Tubulin/metabolism , Acetylation/drug effects , Cell Membrane/metabolism , Cells, Cultured , Cytosol/metabolism , Detergents/chemistry , Endothelial Cells/drug effects , Golgi Apparatus/metabolism , HSP90 Heat-Shock Proteins/metabolism , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydroxamic Acids/pharmacology , Intracellular Membranes/chemistry , Intracellular Membranes/metabolism , Membrane Microdomains/metabolism , Microtubules/drug effects , Nocodazole/pharmacology , Paclitaxel/pharmacology , Phosphorylation/drug effects , Phosphorylation/physiology , Protein Binding/drug effects , Protein Binding/physiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transfection , Tubulin/genetics , Umbilical Veins/cytology , trans-Golgi Network/metabolismABSTRACT
OBJECTIVES: To evaluate the ability of a new apparatus (Dipsys 25, Société SGN, Bagnols sur Cèze, France) to disinfect biomedical waste, including both potentially infectious agents and the normal saprophytic flora of the waste. METHODS: Disinfection was assessed using standard methods (reference strains were fixed on reference carriers according to the French AFNOR methods) and nonstandard assays. Assays in conditions of hospital use, evaluations of bacterial survival during storage, sporicidal effect, and spore survival during storage were performed in parallel. Finally, bactericidal effect in extreme conditions (association of high contamination and high bacterial protection conditions) was tested with normal fecal flora. Bacterial counts were performed after treatment by the apparatus and without treatment (controls). All tests were carried out in triplicate. RESULTS: In all treated carriers, a bacterial population decrease of at least 5 log10 was obtained. Assays performed in hospital-use conditions did not show any bacterial growth. Concerning the evaluation of sporicidal effect and spore revival during conservation, a minimum reduction of 5 log10 was observed in all assays performed, without survival. Finally, concerning assays in extreme conditions, the decrease of bacterial population was between 5 log10 and 10 log10 for vegetative anaerobes of normal fecal flora. CONCLUSION: Under our study conditions, the study apparatus reduced the tested microbial populations by a minimal factor of 5 log10. The main advantage of the apparatus is the opportunity to treat contaminated waste inside hospital wards, at the point of initial collection, without pulverization, by nonspecialized staff.
