Annexins isolation

Annexins isolated from chondroblast matrix vesicles may be reconstituted with phospholipids to form calcium ion channels in the complete absence of Ca2+ ions. Indeed, annexin V has domains that directly bind calcium ions glutamate and aspartate residues provide the ion binding site (EF-hand domains). Figure 9.6b illustrates putative annexin V channels that mediate an influx of Ca2+ ions into artificial bilayers and liposomes (detectable with a calcium-sensitive fluorescent dye). These in vitro annexin Ca2+ channels, and also the Ca2+ influx into matrix vesicles in cell culture and in vivo, are blocked by Zn2+ ions, or a derivative of 1,4-benzothiazepine (inhibitor K201). 

Wright, J.F., Kurosky, A., Pryzdial, E.L., and S. Wasi, 1995, Host cellular annexin II is associated with cytomegalovirus particles isolated from cultured human fibroblasts. J Virol. 69(8) 4784-91. 

The mitomycins are a family of aziridine-containing antibiotics isolated from Streptomyces lavendulae. One of these antibiotics, mitomycin C (MMC), is an antitumor antibiotic frequently used for the treatment of breast, lung, stomach, intestinal, testicular, cholioepithelial, seminal and oral carcinoma [18-20]. When human oral squamous cell carcinoma cell lines (HSC-2, HSC-4) were treated with MMC, the viable cell number was dose-dependently reduced (CC50 of MMC against HSC-2, HSC-3 and HSC-4 cells 3.5, 9.7 and 18 xM, respectively, determined 24 hours after treatment). The rapid decline of polyamines (measured at 3 hours after MMC treatment) was observed prior to the expression of early apoptosis markers such as the production of annexin-positive cells and caspase activation (Table 1) [21]. The interactions... 

Josic D and Lim Y-P. Application of high-performance membrane chromatography for separation of annexins from the plasma membranes of liver and isolation of monospecific polyclonal antibodies. J. Chromatogr. B 1994 662 217-226. 

Isolated nuclei, cells with severely damaged membranes, and very late apoptotic cells stain rapidly and intensely with PI and may not bind annexin V. Stainability of DNA with PI in isolated nuclei is stoichiometric and therefore their frequency histograms of DNA content may have pattern characteristic of the cell-cycle distribution. 

Interpretation of the data is complicated by the presence of nonapoptotic cells with damaged membranes. Such cells may have phosphatidylserine exposed on plasma membrane and, therefore, similar to apoptotic cells, bind annexin V. Mechanical disaggregation of tissues to isolate individual cells extensive use of proteolytic enzymes to disrupt cell aggregates, remove adherent cells from cultures, or to isolate cells from tissue mechanical removal of the cells from tissue culture flasks (e.g., by a rubber policeman) and cell electroporation, may affect the binding of annexin V. Such treatments, therefore, may introduce experimental bias in subsequent analysis of apoptosis by this method. 

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