Phosphatidylserine synthase 2 and phosphatidylserine decarboxylase are essential for aminophospholipid synthesis in Trypanosoma brucei.

Phosphatidylethanolamine (PE) and phosphatidylserine (PS) are ubiquitously expressed and metabolically interconnected glycerophospholipids in eukaryotes and prokaryotes. In Trypanosoma brucei, PE synthesis has been shown to occur mainly via the Kennedy pathway, one of the three routes leading to PE synthesis in eukaryotes, while PS synthesis has not been studied experimentally. We now reveal the importance of T. brucei PS synthase 2 (TbPSS2) and T. brucei PS decarboxylase (TbPSD), two key enzymes involved in aminophospholipid synthesis, for trypanosome viability. By using tetracycline-inducible down-regulation of gene expression and in vivo and in vitro metabolic labeling, we found that TbPSS2 (i) is necessary for normal growth of procyclic trypanosomes, (ii) localizes to the endoplasmic reticulum and (iii) represents the unique route for PS formation in T. brucei. In addition, we identified TbPSD as type I PS decarboxylase in the mitochondrion and found that it is processed proteolytically at a WGSS cleavage site into a heterodimer. Down-regulation of TbPSD expression affected mitochondrial integrity in both procyclic and bloodstream form trypanosomes, decreased ATP production via oxidative phosphorylation in procyclic form and affected parasite growth.

Wave-mixing circular dichroism detector for chiral liquid chromatography.

A circular dichroism (CD) detector based on laser four-wave mixing (FWM) is demonstrated using separate injections of analyte enantiomers onto a standard silica-based microbore high-performance liquid chromatography (HPLC) column. Using the chiral column, a preliminary "detected" mass detection limit of 180 pg is determined inside a laser probe volume of 200 pL, corresponding to a circular dichroism detection limit, DeltaA, of 2.2 x 10(-5) for (-) camphorquinone. Detection sensitivity levels are dramatically improved when our FWM-CD detector is interfaced to a microbore system due to the lower mobile-phase flow rates and the smaller sample concentrations required for the analysis. Using the microbore column, a preliminary circular dichroism detection limit, DeltaA, of 1.6 x 10(-6) and a preliminary concentration detection limit of 4.1 x 10(-4) M are determined for camphorquinone. This corresponds to a "detected" mass detection limit of 33 pg for the chiral compound. Laser wave mixing offers better detection limits than conventional circular dichroism detection methods and, hence, offers many promising applications.