A Novel Non-Lens βγ−Crystallin and Trefoil Factor Complex from Amphibian Skin and Its Functional Implications

In vertebrates, non-lens betagamma-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive.A naturally existing 72-kDa complex of non-lens betagamma-crystallin (alpha-subunit) and trefoil factor (beta-subunit), named betagamma-CAT, was identified from frog Bombina maxima skin secretions. Its alpha-subunit and beta-subunit (containing three trefoil factor domains), with a non-covalently linked form of alphabeta(2), show significant sequence homology to ep37 proteins, a group of non-lens betagamma-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. betagamma-CAT showed potent hemolytic activity on mammalian erythrocytes. The specific antiserum against each subunit was able to neutralize its hemolytic activity, indicating that the two subunits are functionally associated. betagamma-CAT formed membrane pores with a functional diameter about 2.0 nm, leading to K(+) efflux and colloid-osmotic hemolysis. High molecular weight SDS-stable oligomers (>240-kDa) were detected by antibodies against the alpha-subunit with Western blotting. Furthermore, betagamma-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of betagamma-CAT (25-50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC(50) 10 nM) and apoptosis. betagamma-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. Bafilomycin A1 (a specific inhibitor of the vacuolar-type ATPase) and nocodazole (an agent of microtuble depolymerizing), while inhibited betagamma-CAT induced vacuole formation, significantly inhibited betagamma-CAT induced cell detachment, suggesting that betagamma-CAT endocytosis is important for its activities.These findings illustrate novel cellular functions of non-lens betagamma-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals

Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2α. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-α. This was concomitant with increased IL-8 synthesis and cPLA2α activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-β-cyclodextrin induced further cPLA2α activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-α-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2α and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-α-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis

Using soil water depletion to measure spatial distribution of root activity in oil palm (Elaeis guineensis Jacq.) plantations

Knowledge of where roots are active is crucial for efficient management of nutrients in tree crops but measurement of root activity is problematic. Measurement using soil water depletion is an approach that has not been tested in a humid climate. We hypothesized that the three dimensional distribution of root activity of a tree crop in the humid tropics a) can be determined by measuring soil water depletion during rain-free periods, and b) is influenced by environment (soil type and climate) and surface management. A field study was carried out in which soil water content was measured and water uptake calculated (by difference between soil water content at beginning and end of rain-free periods) for different surface management zones and depths (0.1 m intervals to 1.6 m depth) under oil palm (Elaeis guineensis Jacq.) at a loam-clay site and a sandy site. Significant differences were measured between sites and between surface management zones at each site. At both sites water uptake was highest under the weeded zone close to the palm stem, slightly lower under the zone where pruned fronds are placed, and lowest under the path used for removing harvested fruit. Vertical distribution of root activity differed between the sites, with higher activity near the surface at the finer textured site. Total water uptake values were lower than estimates of evapotranspiration made using climate data. The difference was probably largely due to water uptake from deeper than 1.6 m. This study showed that the spatial distribution of tree root activity in a humid climate could be quantified using a relatively simple method