SummaryThe assembly of vital reactive iron-sulfur (Fe-S) cofactors in eukaryotes is mediated by proteins inherited from the original mitochondrial endosymbiont. Uniquely among eukaryotes, however, Entamoeba and Mastigamoeba lack such mitochondrial-type Fe-S cluster assembly proteins and possess instead an analogous bacterial-type system acquired by lateral gene transfer. Here we demonstrate, using immunomicroscopy and biochemical methods, that beyond their predicted cytosolic distribution the bacterial-type Fe-S cluster assembly proteins NifS and NifU have been recruited to function within the relict mitochondrial organelles (mitosomes) of Entamoeba histolytica. Both Nif proteins are 10-fold more concentrated within mitosomes compared with their cytosolic distribution suggesting that active Fe-S protein maturation occurs in these organelles. Quantitative immunoelectron microscopy showed that amoebal mitosomes are minute but highly abundant cellular structures that occupy up to 2% of the total cell volume. In addition, protein colocalization studies allowed identification of the amoebal hydroperoxide detoxification enzyme rubrerythrin as a mitosomal protein. This protein contains functional Fe-S centres and exhibits peroxidase activity in vitro. Our findings demonstrate the role of analogous protein replacement in mitochondrial organelle evolution and suggest that the relict mitochondrial organelles of Entamoeba are important sites of metabolic activity that function in Fe-S proteinmediated oxygen detoxification.

A rapid and sensitive method for the simultaneous confirmatory analysis of three forensic most relevant cannabinoids, Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH), by means of high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) in human plasma was developed and fully validated. Sample clean-up was performed by automated silica-based solid-phase extraction and the separation was carried out using a PhenylHexyl column (50 x 2 mm i.d., 3 micro m) and acetonitrile-5 mM ammonium acetate gradient elution. Data were acquired with an API 3000 LC/MS/MS system equipped with a turboionspray interface and triple quadrupole mass analyzer using positive electrospray ionization and multiple reaction monitoring. Two MS/MS transitions for each substance were monitored and deuterated analogues of analytes were used as internal standards for quantitation. The limit of quantitation was 0.8 ng ml(-1) for THC, 0.8 ng ml(-1) for 11-OH-THC and 4.3 ng ml(-1) for THC-COOH and linearity with a correlation coefficient r(2) = 0.999 was achieved up to 100 ng ml(-1) for THC and 11-OH-THC and 500 ng ml(-1) for THC-COOH. The limits of detection were 0.2 ng ml(-1) for THC, 0.2 ng ml(-1) for 11-OH-THC and 1.6 ng ml(-1) for THC-COOH. The developed LC/MS/MS method was also successfully used for the determination of THC-COOH-glucuronide, the phase II metabolite of THC-COOH.

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