Effect of Pseudomonas aeruginosa (MTCC 1688) on the tissue phosphatases activity on Macrobrachium rosenbergii (De Man).

A time course study on the endotoxin toxicity of the gram negative bacteria, Pseudomonas aeruginosa MTCC 1688 on the tissue phosphatases activity on the giant freshwater prawn, Macrobrachium rosenbergii was conducted. The results revealed marked elevation of both acid and alkaline phosphatase activity in the haemolymph and body muscle. The hepatopancreas showed reduced phosphatase activity compared to control. The enzymes, being non-specific in action and particularly the acid phosphatase being of lysosomal origin, their increase in muscle and haemolymph has pathogenic significance in the inoculum treated prawns.

Cell-free expression and functional reconstitution of CALM in clathrin assembly

Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the protein and lipid. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). In order to better understand a possible role of post-translational modification of CALM (clathrin assembly protein lymphoid myeloid), the homologue of AP180, in the assembly of CCVs, CALM was expressed in the cell-free reticulocyte translation system that is capable of carrying out post-translational modification. The apparent molecular weight of the expressed recombinant CALM was estimated as 105 kD. Alkaline phosphatase treatment of CALM resulted in a mobility shift on SDS-PAGE. We found that CALM was associated with the proteins harboring SH3 domain, promote assembly of clathrin triskelia into clathrin cage and bound to the preformed clathrin cage. CALM was also proteolyzed by caspase 3 and calpain but not by caspase 8. These results indicated that the post-translationally modified CALM, expressed in the eukaryotic cell-free reticulocyte translation system was able to mediate the assembly of clathrin and the coated-vesicle formation.