Caveolae-mediated endocytosis of the glucosaminoglycan-interacting adipokine tartrate resistant acid phosphatase 5a in adipocyte progenitor lineage cells

Adipogenesis depends on growth factors controlling proliferation/differentiation of mesenchymal stem cells (MSCs). Membrane binding and endocytosis of growth factors are often coupled to receptor activation and downstream signaling leading to specific cellular responses. The novel adipokine tartrate-resistant acid phosphatase (TRAP) 5a exhibits a growth factor-like effect on MSCs and pre-adipocytes and induces hyperplastic obesity in vivo. However its molecular interaction with pre-adipocytes remains unknown. Therefore, this study aimed to investigate membrane interaction of TRAP and its endocytosis routes in pre-adipocytes. Confocal and/or electron microscopy were used to detect TRAP in untreated or TRAP 5a/b treated pre-adipocytes under conditions that allow or inhibit endocytosis in combination with co-staining of endocytotic vesicles. TRAP interaction with heparin/heparan sulfate was verified by gel filtration. It could be shown that TRAP 5a, but not 5b, binds to the membrane of pre-adipocytes where it co-localizes with heparin-sulfate proteoglycan glypican-4. Also in vitro, TRAP 5a exhibited affinity for both heparin and heparan sulfate with heparin inhibiting its enzyme activity. Upon caveolae-mediated endocytosis of saturating levels of TRAP 5a, TRAP 5a co-localized intracellularly with glypican-4 and late endosomal marker Rab-7 positive vesicles. The protein was also located in multivesicular bodies (MVBs) but did not co-localize with lysosomal marker LAMP-1. TRAP 5a endocytosis was also detectable in pre-osteoblasts, but not fibroblasts, embryonic MSCs or mature adipocytes. These results indicate that TRAP 5a exhibits binding to cell surface, endocytosis and affinity to glucosaminoglycans (GAGs) in pre-adipocyte and pre-osteoblast lineage cells in a manner similar to other heparin-binding growth factors.     

Monodansylcadaverine inhibits cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin on cultured rat embryonic fibroblast cells

The mechanism of action of Vibrio parahaemolyticus thermostable direct hemolysin (TDH) on cultured cells still remains unclear. We show that addition of osmotic stabilizers, such as polyethylene glycol and dextran, could not protect cultured rat embryonic fibroblast cells (Rat-1) against cytotoxicity induced by TDH, unlike their protection against the hemolytic activity of TDH. By contrast, 100 microM monodansylcadaverine, as well as the presence of 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in medium, protected the cells against cytotoxicity of TDH. Binding of TDH to Rat-1 cells and intracellular localization of TDH were affected by monodansylcadaverine and EGTA as analyzed by flow cytometry and confocal microscopy. On the hemolytic activity of TDH, monodansylcadaverine and EGTA had no effect. These results suggest that the mechanism of cytotoxicity of TDH on Rat-1 cells was different from that of hemolytic activity of TDH on red blood cells.     

自噬的检测方法及评述

细胞自噬是广泛存在于真核细胞内的一种降解途径,在机体发育过程中、在生理和病理状况下都起重要作用。近年,自噬成为热点研究领域,但少有论文深入分析不同检测方法得来的数据在研究中的不同意义,说明不同检测方法所获的结果的优缺点和适用条件。本文拟对哺乳动物细胞自噬检测方法进行归类、介绍及评述,旨在为研究工作中自噬检测方法的选择和研究结果的解释起帮助作用。     

Impairment of the cytotoxic and oxidative activities of 7 beta-hydroxycholesterol and 7-ketocholesterol by esterification with oleate

Atherosclerosis involves inflammatory processes, as well as cytotoxic and oxidative reactions. In atherosclerotic plaques, these phenomena are revealed by the presence of dead cells, oxidized lipids, and oxidative DNA damage, but the molecules triggering these events are still unknown. As 7 beta-hydroxycholesterol and 7-ketocholesterol, which are present at elevated concentrations in atherosclerotic lesions, are strongly cytotoxic and pro-oxidative, their effects were determined on cell death, superoxide anion and nitric oxide production, lipid peroxidation, and oxidative DNA damage. 7-Ketocholesterol- and 7 beta-hydroxycholesterol-induced cell death leads to a loss of mitochondrial potential, to increased permeability to propidium iodide, and to morphological nuclear changes (swelling, fragmentation, and/or condensation of nuclei). These effects are preceded by the formation of cytoplasmic monodansylcadaverine-positive structures and are associated with a rapid enhancement of cells overproducing superoxide anions, a decrease in cells producing nitric oxide, lipid peroxidation (formation of malondialdehyde and 4-hydroxynonenal adducts, low ratio of [unsaturated fatty acids]/[saturated fatty acids]) as well as oxidative DNA damage (8-oxoguanine formation). Noteworthy, none of the cytotoxic features previously observed with 7 beta-hydroxycholesterol and 7-ketocholesterol were noted with cholesterol, 7 beta-hydroxycholesteryl-3-oleate and 7-ketocholesteryl-3-oleate, with the exception of a slight increase in superoxide anion production with 7 beta-hydroxycholesteryl-3-oleate. This finding supports the theory that 7 beta-hydroxycholesterol and 7-ketocholesterol could induce cytotoxic and oxidative processes observed in atherosclerotic lesions and that esterification of these compounds may contribute to reducing atherosclerosis progression.