Trypanosoma cruziProlyl Oligopeptidase Tc80 Is Involved in Nonphagocytic Mammalian Cell Invasion by Trypomastigotes

Trypanosoma cruziis an intracellular protozoan parasite able to invade a wide variety of mammalian cells. To have access to the target organs/cells, the parasite must cross the basal laminae and the extracellular matrix (ECM). We previously characterized an 80-kDa proteinase (Tc80) secreted by the infective trypomastigotes that hydrolyzes native collagens and might be involved in infection by degrading ECM components. Here, we present evidence indicating a role for Tc80 in the invasion of nonphagocytic cells. Tc80 was classified as a member of the prolyl oligopeptidase (POP) family of serine proteases and was also found to hydrolyze fibronectin. Selective inhibitors for POP Tc80 were synthesized that blocked parasite entry into cells. Blockage occurred when trypomastigotes were preincubated with irreversible inhibitors but not after host cell preincubation, and the blockage correlated with inhibition of POP Tc80 activity in treated parasites. These data and the enzyme location inside a vesicular compartment close to the flagellar pocket, a specialized domain in endocytosis/exocytosis, strongly suggest a role for POP Tc80 in the maturation of parasite protein(s) and/or, after secretion, in a local action on parasite or host cell/ECM components required for invasion.

Immunolocalization of NA+,K+-ATPase in the Branchial Cavity During the Early Development of the European Lobster Homarus Gammarus (Crustacea, Decapoda)

We examined the ontogeny of the osmoregulatory sites of the branchial cavity in embryonic and early postembryonic stages of the European lobster Homarus gammarus through transmission electron microscopy, immunofluorescence microscopy, and immunogold electron microscopy using a monoclonal antibody IgGα5 raised against the avian α-subunit of the Na+,K+-ATPase. In mid-late embryos, Na+,K+-ATPase was located along the pleurites and within the epipodite buds. In late embryos just before hatching, the enzyme was confined to the epipodite epithelia. After hatching, slight differentiations of ionocytes occured in the epipodites of larval stages. Na+,K+-ATPase was also located in the ionocytes of the epipodites of larvae exposed to seawater (35.0‰) and to dilute seawater (22.1 ‰). After metamorphosis, the inner-side branchiostegite epithelium appeared as an additional site of enzyme location in postlarvae held in dilute seawater. Within the ionocytes, Na+,K+-ATP-ase was mostly located along the basolateral infoldings. These observations are discussed in relation to the physiological shift from osmoconforming larvae to slightly hyper-regulating (in dilute seawater) postmetamorphic stages. The acquisition of the ability to hyper-osmo-regulate probably originates from the differentiation, on the epipodites and mainly along the branchiostegites, of ionocytes that are the site of ion pumping as evidenced by the location of Na+,K+-ATPase. (J Histochem Cytochem 49:1013–1023, 2001)

Mimicked translocation of glucose and glucose 6-phosphate with artificial enzyme membranes

An approach to the mechanism which may govern the behaviour of biological compartmentalized systems is presented. Artificial enzyme membranes with immobilized glucose oxidase, invertase or hexokinase were used to separate two compartments of a specially designed diffusion cell. Asymmetry in volume, hydrodynamic conditions and enzyme location was purposely chosen in order to create situations which could not be obtained with an enzyme free in solution, and was then used to tentatively mimic situations existing in vivo. Experiments were conducted and a translocation effect of H2O2, glucose and glucose 6-phosphate was obtained. A theoretical analysis taking into account the different identified parameters of the system was elaborated.