Biomedical subjects

Dan Ye

Publications and source records attributed to Dan Ye.

21 records · Page 2Linked to original sources

PubMed2002

Cytochrome p-450 epoxygenase metabolites of docosahexaenoate potently dilate coronary arterioles by activating large-conductance calcium-activated potassium channels.

Diets enriched in docosahexaenoic acid, a major n-3 fatty acid in fish oil, have hypotensive properties. One mechanism that can lower blood pressure is the direct dilation of arterioles by docosahexaenoic metabolites. Vascular endothelium contains cytochrome P-450 epoxygenases that transform the n-6 fatty acid arachidonate into epoxyeicosatrienoic acids (EETs), potent dilators of coronary arterioles and activators of large-conductance calcium-activated potassium (BK(Ca)) channels. To test whether analogous activations occur for docosahexaenoate, we compared the potency of docosahexaenoate and its five cytochrome P-450 epoxygenase metabolites, epoxydocosapentaenoates (EDPs), in dilating porcine coronary arterioles (<135 microm in diameter) precontracted with endothelin. The five EDP regioisomers had dilation EC(50) values ranging from 0.5 to 24 pM (n = 5-6). In contrast, the EDP hydrolysis product 13,14-dihydroxydocosapentaenoic acid (13,14-DHDP) had an EC(50) value of 30 +/- 22 nM (n = 7), whereas docosahexaenoate only dilated vessels at > or =1.0 microM (n = 7). Using patch-clamp techniques in the inside-out configuration, we determined that the 13,14-EDP regioisomer potently activated (EC(50) value of 6.6 +/- 0.6 pM; n = 5) BK(Ca) channels in myocytes from the porcine coronary arterioles. Moreover, 13,14-EDP potently activated BK(Ca) channels in myocytes from rat coronary small arteries (150-300 microm in diameter); with an EC(50) value of 2.2 +/- 0.6 pM (n = 7), 13,14-EDP was 1000-fold more potent than EETs in activating BK(Ca) channels. We conclude that EDPs potently dilate coronary microvessels and are the most potent fatty epoxides known to activate BK(Ca) channels in coronary smooth muscle cells. Both actions may contribute to the hypotensive effects of dietary fish oils.

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PubMed2002

Fluorescence resonance energy transfer microscopy of the Helicobacter pylori vacuolating cytotoxin within mammalian cells.

The Helicobacter pylori vacuolating cytotoxin (VacA) binds and enters mammalian cells to induce cellular vacuolation. To investigate the quaternary structure of VacA within the intracellular environment where toxin cytotoxicity is elaborated, we employed fluorescence resonance energy transfer (FRET) microscopy. HeLa cells coexpressing full-length and truncated forms of VacA fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) were analyzed for FRET to indicate direct associations. These studies revealed that VacA-CFP and VacA-YFP interact within vacuolated cells, supporting the belief that monomer associations at an intracellular site are important for the toxin's vacuolating activity. In addition, the two fragments of proteolytically nicked VacA, p37 and p58, interact when coexpressed within mammalian cells. Because p37 and p58 function in trans when expressed separately within mammalian cells, these data suggest that the mechanism by which these two fragments induce vacuolation requires direct association. FRET microscopy also demonstrated interactions between mutant forms of VacA, as well as wild-type VacA with mutant forms of the toxin within vacuolated cells. Finally, a dominant-negative form of the toxin directly associates with wild-type VacA in cells where vacuolation was not detectable, suggesting that the formation of complexes comprising wild-type and dominant-negative forms of toxin acts to block intracellular toxin function.

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PubMed2002

Plasma membrane cholesterol modulates cellular vacuolation induced by the Helicobacter pylori vacuolating cytotoxin.

The Helicobacter pylori vacuolating cytotoxin (VacA) induces the degenerative vacuolation of mammalian cells both in vitro and in vivo. Here, we demonstrate that plasma membrane cholesterol is essential for vacuolation of mammalian cells by VacA. Vacuole biogenesis in multiple cell lines was completely blocked when cholesterol was extracted selectively from the plasma membrane by using beta-cyclodextrins. Moreover, increasing plasma membrane cholesterol levels strongly potentiated VacA-induced vacuolation. In contrast, inhibiting de novo biosynthesis of cholesterol with lovastatin or compactin had no detectable effect on vacuolation. While depletion of plasma membrane cholesterol has been shown to interfere with both clathrin-mediated endocytosis and caveola-dependent endocytosis, neither of these two internalization pathways was found to be essential for vacuolation of cells by VacA. Depleting plasma membrane cholesterol attenuated the entry of VacA into HeLa cells. In addition, beta-cyclodextrin reagents blocked vacuolation of cells that were either preloaded with VacA or had VacA directly expressed within the cytosol. Collectively, our results suggest that plasma membrane cholesterol is important for both the intoxication mechanism of VacA and subsequent vacuole biogenesis.

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