Specific and reversible inhibition of entamoeba histolytica cysteine proteinase activities by Zn²+: Implications for adhersion and cell damage1

Background. Cysteine-proteinases are thought to play an important role in E. histolytica pathogenicity. Although effective blockage of proteolytic activities can be obtained with sereveral known inhibitors, the high cellular toxicity of most of the inhibitors precludes experimentation with live cells or animal models. Specific cysteine-proteinase inhibitors that could be utilized in studies of virulence are of great need in the field of amebiasis. Methods. Cysteine-proteinase activities were determined in trophozoit lysates by azocasein degradation and after PAGE and gelatin zymograms. Inhibition of the activities was assessed in the presence of 0.01-2.5 mM concentrations of fivalent cations of the IIB and VIII series such as Zn, Cd, Hg, Ni, and Co. Reversibility was induced with 25 mM L-cysteine or 50 mM L-histidine and by metal chelation with 5 mM phenantroline. The inhibitory effect of ZnCI2 was tested with live cells in fibronectin-biding and cytotoxicity assays. Results. ZnCI2 specifically inhibited cysteine-proteinase activities in trophozoite lysates in a concentration-dependent manner. Additionally, 1.0-2.5 mM ZnCI2 bloked proteolysis in more than 70 percent. This inhibition was completely reverted by L-cysteine, L-histidine, or phenantroline. Similar results were obtained by analyzing indivual cysteine-proteinase activities separated in gelatin zymograms. At these concentrations, ZnCI2 significanty interfered with trophozoit adhesion, thus making amebas deficient in substrate degradation and cell damage. Conclusions. ZnCI2 is effective inhibitor of amebic cysteine-proteinases. Its low toxicity at relative high concentrations, high solubility, and low cost make it adequate for live cell experimentation and animal models of amebic virulence

Myosin I interactions with actin filaments and trans-golgi-derived vesicles in MDCK cell monolayers

In MDCK cell cultured monolayers, as well as in natural and other cultured epithelia, the proper organization of the actin filament ring, tethered to the plasma memebrane at the zonula adhaerens, is apparently necessary for their functioning as a transporting epithelium. It has been proposed that actin filaments, in conjunction with motor proteins, could provide the structural basis that regulates the tight junction (TJ) sealing capacity as well as the transport of memebrane-tagged proteins required for cell polarization. To test this hypothesis, the authors analyzed the localization and possible association ot the actin binding motor protein myosin I with actin filaments during changes in the actin ring position and organization, and also with tran-Golgi-derived vesicle. Modifications of the ring were induced subjecting the cells to external Ca²+ switch), or by treatment with drugs known to depolymerize actin filament (cytochalasin D, CD). The distribution of myosin I and actin, both in intact cells and in cellular fractions, was monitored using heterlogous cross-reacting antibodies and phalloidin. The authors identified an isoform of myosin I of approximately 110-125 KDa, homologus to myosin IB of Acanthamoeba, a fraction of wich colocalized with the peripheral actin ring. The association seems transient as, once the ring retracted as result of Ca²+ depletion, or became disroganized by CD, myosin not longer colocalized with the actin fibers but appeared dispersed in the cytoplasm. Furthermore, a signficant fraction of the total myosin I in the cell was associated to Golgi-derived vesicles which could also associate in vitro with actin filaments. The authors' data support, then, the participation of myosin I, in association with actin filaments, in vesicle translocation to and from the cell membrane as proposed for natural epithelia, and provide a further insigh into the structural organization that maintains epithelial cell polatiry in cultured monolayers