Lipid metabolism in Trypanosoma brucei

Trypanosoma brucei membranes consist of all major eukaryotic glycerophospholipid and sphingolipid classes. These are de novo synthesized from precursors obtained either from the host or from catabolised endocytosed lipids. In recent years, substantial progress has been made in the molecular and biochemical characterisation of several of these lipid biosynthetic pathways, using gene knockout or RNA interference strategies or by enzymatic characterization of individual reactions. Together with the completed genome, these studies have highlighted several possible differences between mammalian and trypanosome lipid biosynthesis that could be exploited for the development of drugs against the diseases caused by these parasites.

Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modelling

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a'_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.

Characterization of an ADP-ribosyltransferase toxin (AexT) from Aeromonas salmonicida subsp. salmonicida

An ADP-ribosylating toxin named Aeromonas salmonicida exoenzyme T (AexT) in A. salmonicida subsp. salmonicida, the etiological agent of furunculosis in fish, was characterized. Gene aexT, encoding toxin AexT, was cloned and characterized by sequence analysis. AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of Pseudomonas aeruginosa and to the YopE cytotoxin of different Yersinia species. The aexT gene was detected in all of the 12 A. salmonicida subsp. salmonicida strains tested but was absent from all other Aeromonas species. Recombinant AexT produced in Escherichia coli possesses enzymatic ADP-ribosyltransferase activity. Monospecific polyclonal antibodies directed against purified recombinant AexT detected the toxin produced by A. salmonicida subsp. salmonicida and cross-reacted with ExoS and ExoT of P. aeruginosa. AexT toxin could be detected in a wild type (wt) strain of A. salmonicida subsp. salmonicida freshly isolated from a fish with furunculosis; however, its expression required contact with RTG-2 rainbow trout gonad cells. Under these conditions, the AexT protein was found to be intracellular or tightly cell associated. No AexT was found when A. salmonicida subsp. salmonicida was incubated in cell culture medium in the absence of RTG-2 cells. Upon infection with wt A. salmonicida subsp. salmonicida, the fish gonad RTG-2 cells rapidly underwent significant morphological changes. These changes were demonstrated to constitute cell rounding, which accompanied induction of production of AexT and which led to cell lysis after extended incubation. An aexT mutant which was constructed from the wt strain with an insertionally inactivated aexT gene by allelic exchange had no toxic effect on RTG-2 cells and was devoid of AexT production. Hence AexT is directly involved in the toxicity of A. salmonicida subsp. salmonicida for RTG-2 fish cells.

Characterization of a novel CYP19A1 (aromatase) R192H mutation causing virilization of a 46,XX newborn, undervirilization of the 46,XY brother, but no virilization of the mother during pregnancies.

BACKGROUND P450 aromatase (CYP19A1) is essential for the biosynthesis of estrogens from androgen precursors. Mutations in the coding region of CYP19A1 lead to autosomal recessive aromatase deficiency. To date over 20 subjects have been reported with aromatase deficiency which may manifest during fetal life with maternal virilization and virilization of the external genitalia of a female fetus due to low aromatase activity in the steroid metabolizing fetal-placental unit and thus high androgen levels. During infancy, girls often have ovarian cysts and thereafter fail to enter puberty showing signs of variable degree of androgen excess. Moreover, impact on growth, skeletal maturation and other metabolic parameters is seen in both sexes. OBJECTIVE AND HYPOTHESIS We found a novel homozygous CYP19A1 mutation in a 46,XX girl who was born at term to consanguineous parents. Although the mother did not virilize during pregnancy, the baby was found to have a complex genital anomaly at birth (enlarged genital tubercle, fusion of labioscrotal folds) with elevated androgens at birth, normalizing thereafter. Presence of 46,XX karyotype and female internal genital organs (uterus, vagina) together with biochemical findings and follow-up showing regression of clitoral hypertrophy, as well as elevated FSH suggested aromatase deficiency. Interestingly, her older brother presented with mild hypospadias and bilateral cryptorchidism and was found to carry the same homozygous CYP19A1 mutation. To confirm the clinical diagnosis, genetic, functional and computational studies were performed. METHODS AND RESULTS Genetic analysis revealed a homozygous R192H mutation in the CYP19A1 gene. This novel mutation was characterized for its enzymatic activity (Km, Vmax) in a cell model and found to have markedly reduced catalytic activity when compared to wild-type aromatase; thus explaining the phenotype. Computational studies suggest that R192H disrupts the substrate access channel in CYP19A1 that may affect binding of substrates and exit of catalytic products. CONCLUSION R192H is a novel CYP19A1 mutation which causes a severe phenotype of aromatase deficiency in a 46,XX newborn and maybe hypospadias and cryptorchidism in a 46,XY, but no maternal androgen excess during pregnancy.

Electrophoretically mediated microanalysis for characterization of the enantioselective CYP3A4 catalyzed N-demethylation of ketamine

Execution of an enzymatic reaction performed in a capillary with subsequent electrophoretic analysis of the formed products is referred to as electrophoretically mediated microanalysis (EMMA). An EMMA method was developed to investigate the stereoselectivity of the CYP3A4-mediated N-demethylation of ketamine. Ketamine was incubated in a 50 μm id bare fused-silica capillary together with human CYP3A4 Supersomes using a 100 mM phosphate buffer (pH 7.4) at 37°C. A plug containing racemic ketamine and the NADPH regenerating system including all required cofactors for the enzymatic reaction was injected, followed by a plug of the metabolizing enzyme CYP3A4 (500 nM). These two plugs were bracketed by plugs of incubation buffer to ensure proper conditions for the enzymatic reaction. The rest of the capillary was filled with a pH 2.5 running buffer comprising 50 mM Tris, phosphoric acid, and 2% w/v of highly sulfated γ-cyclodextrin. Mixing of reaction plugs was enhanced via application of -10 kV for 10 s. After an incubation of 8 min at 37°C without power application (zero-potential amplification), the capillary was cooled to 25°C within 3 min followed by application of -10 kV for the separation and detection of the formed enantiomers of norketamine. Norketamine formation rates were fitted to the Michaelis-Menten model and the elucidated values for V(max) and K(m) were found to be comparable to those obtained from the off-line assay of a previous study.

Synthesis and Biochemical Characterization of Tricyclothymidine Triphosphate (tc-TTP)

Tricyclo-DNA (tc-DNA) is a conformationally restricted oligonucleotide analogue that exhibits promising properties as a robust antisense agent. Here we report on the synthesis and biochemical characterization of tc-TTP, the triphosphate of a tc-DNA nucleoside containing the base thymine. Tc-TTP turned out to be a substrate for the Vent (exo−) DNA polymerase, a polymerase that allows for multiple incorporations of tc-T nucleotides under primer extension reaction conditions. However, the substrate acceptance is rather low, as also observed for other sugar-modified analogues. Tc-TTP and tc-nucleotide-containing templates do not sustain enzymatic polymerization under physiological conditions; this indicates that tc-DNA-based antisense agents will not enter natural metabolic pathways that lead to long-term toxicity.