The Sphingolipid Biosynthetic Pathway Is a Potential Target for Chemotherapy against Chagas Disease

The protozoan parasite Trypanosoma cruzi is the causative agent of human Chagas disease, for which there currently is no cure. The life cycle of T. cruzi is complex, including an extracellular phase in the triatomine insect vector and an obligatory intracellular stage inside the vertebrate host. These phases depend on a variety of surface glycosylphosphatidylinositol-(GPI-) anchored glycoconjugates that are synthesized by the parasite. Therefore, the surface expression of GPI-anchored components and the biosynthetic pathways of GPI anchors are attractive targets for new therapies for Chagas disease. We identified new drug targets for chemotherapy by taking the available genome sequence information and searching for differences in the sphingolipid biosynthetic pathways (SBPs) of mammals and T. cruzi. In this paper, we discuss the major steps of the SBP in mammals, yeast and T. cruzi, focusing on the IPC synthase and ceramide remodeling of T. cruzi as potential therapeutic targets for Chagas disease

The Accretion Flows and Evolution of Magnetic Cataclysmic Variables

We have used a model of magnetic accretion to investigate the accretion flows of magnetic cataclysmic variables. Numerical simulations demonstrate that four types of flow are possible: discs, streams, rings and propellers. The fundamental observable determining the accretion flow, for a given mass ratio, is the spin-to-orbital period ratio of the system. If IPs are accreting at their equilibrium spin rates, then for a mass ratio of 0.5, those with Pspin/Porb < 0.1 will be disc-like, those with 0.1 < Pspin/Porb < 0.6 will be stream-like, and those with Pspin/Porb ~ 0.6 will be ring-like. The spin to orbital period ratio at which the systems transition between these flow types increases as the mass ratio of the stellar components decreases. For the first time we present evolutionary tracks of mCVs which allow investigation of how their accretion flow changes with time. As systems evolve to shorter orbital periods and smaller mass ratios, in order to maintain spin equilibrium, their spin-to-orbital period ratio will generally increase. As a result, the relative occurrence of ring-like flows will increase, and the occurrence of disc-like flows will decrease, at short orbital periods. The growing number of systems observed at high spin-to-orbital period ratios with orbital periods below 2h, and the observational evidence for ring-like accretion in EX Hya, are fully consistent with this picture.Comment: Accepted for publication in ApJ. 6 figures - included here at low resolutio

Tamoxifen inhibits the biosynthesis of inositolphosphorylceramide in Leishmania

Previous work from our group showed that tamoxifen, an oral drug that has been in use for the treatment of breast cancer for over 40 years, is active both in vitro and in vivo against several species of Leishmania, the etiological agent of leishmaniasis. Using a combination of metabolic labeling with [3H]-sphingosine and myo-[3H]-inositol, alkaline hydrolysis, HPTLC fractionations and mass spectrometry analyses, we observed a perturbation in the metabolism of inositolphosphorylceramides (IPCs) and phosphatidylinositols (PIs) after treatment of L. amazonensis promastigotes with tamoxifen, with a significant reduction in the biosynthesis of the major IPCs (composed of d16:1/18:0-IPC, t16:0/C18:0-IPC, d18:1/18:0-IPC and t16:0/20:0-IPC) and PIs (sn-1-O-(C18:0)alkyl -2-O-(C18:1)acylglycerol-3-HPO4-inositol and sn-1-O-(C18:0)acyl-2-O-(C18:1)acylglycerol-3-HPO4-inositol) species. Substrate saturation kinetics of myo-inositol uptake analyses indicated that inhibition of inositol transport or availability were not the main reasons for the reduced biosynthesis of IPC and PI observed in tamoxifen treated parasites. An in vitro enzymatic assay was used to show that tamoxifen was able to inhibit the Leishmania IPC synthase with an IC50 value of 8.48 μM (95% CI 7.68–9.37), suggesting that this enzyme is most likely one of the targets for this compound in the parasites

Acidocalcisomes as Calcium- and Polyphosphate-Storage Compartments during Embryogenesis of the Insect Rhodnius prolixus Stahl

BACKGROUND: The yolk of insect eggs is a cellular domain specialized in the storage of reserve components for embryo development. The reserve macromolecules are stored in different organelles and their interactions with the embryo cells are mostly unknown. Acidocalcisomes are lysosome-related organelles characterized by their acidic nature, high electron density and large content of polyphosphate bound to several cations. In this work, we report the presence of acidocalcisome-like organelles in eggs of the insect vector Rhodnius prolixus. METHODOLOGY/PRINCIPAL FINDINGS: Characterization of the elemental composition of electron-dense vesicles by electron probe X-ray microanalysis revealed a composition similar to that previously described for acidocalcisomes. Following subcellular fractionation experiments, fractions enriched in acidocalcisomes were obtained and characterized. Immunofluorescence showed that polyphosphate polymers and the vacuolar proton translocating pyrophosphatase (V-H(+)-PPase, considered as a marker for acidocalcisomes) are found in the same vesicles and that these organelles are mainly localized in the egg cortex. Polyphosphate quantification showed that acidocalcisomes contain a significant amount of polyphosphate detected at day-0 eggs. Elemental analyses of the egg fractions showed that 24.5±0.65% of the egg calcium are also stored in such organelles. During embryogenesis, incubation of acidocalcisomes with acridine orange showed that these organelles are acidified at day-3 (coinciding with the period of yolk mobilization) and polyphosphate quantification showed that the levels of polyphosphate tend to decrease during early embryogenesis, being approximately 30% lower at day-3 compared to day-0 eggs. CONCLUSIONS: We found that acidocalcisomes are present in the eggs and are the main storage compartments of polyphosphate and calcium in the egg yolk. As such components have been shown to be involved in a series of dynamic events that may control embryo growth, results reveal the potential involvement of a novel organelle in the storage and mobilization of inorganic elements to the embryo cells

Venom alkaloids against Chagas disease parasite: search for effective therapies

Chagas disease is an important disease affecting millions of patients in the New World and is caused by a protozoan transmitted by haematophagous kissing bugs. It can be treated with drugs during the early acute phase; however, effective therapy against the chronic form of Chagas disease has yet to be discovered and developed. We herein tested the activity of solenopsin alkaloids extracted from two species of fire ants against the protozoan parasite Trypanosoma cruzi, the aetiologic agent of Chagas disease. Although IC50 determinations showed that solenopsins are more toxic to the parasite than benznidazole, the drug of choice for Chagas disease treatment, the ant alkaloids presented a lower selectivity index. As a result of exposure to the alkaloids, the parasites became swollen and rounded in shape, with hypertrophied contractile vacuoles and intense cytoplasmic vacuolization, possibly resulting in osmotic stress; no accumulation of multiple kinetoplasts and/or nuclei was detected. Overexpressing phosphatidylinositol 3-kinase—an enzyme essential for osmoregulation that is a known target of solenopsins in mammalian cells—did not prevent swelling and vacuolization, nor did it counteract the toxic effects of alkaloids on the parasites. Additional experimental results suggested that solenopsins induced a type of autophagic and programmed cell death in T. cruzi. Solenopsins also reduced the intracellular proliferation of T. cruzi amastigotes in infected macrophages in a concentration-dependent manner and demonstrated activity against Trypanosoma brucei rhodesiense bloodstream forms, which is another important aetiological kinetoplastid parasite. The results suggest the potential of solenopsins as novel natural drugs against neglected parasitic diseases caused by kinetoplastids.Fil: Silva, Rafael C. M. Costa. Universidade Federal do Rio de Janeiro; BrasilFil: Fox, Eduardo G. P.. Universidade Federal do Rio de Janeiro; Brasil. South China Agricultural University; ChinaFil: Gomes, Fabio M.. Universidade Federal do Rio de Janeiro; Brasil. National Institutes of Health; Estados UnidosFil: Feijó, Daniel F.. Universidade Federal do Rio de Janeiro; BrasilFil: Ramos, Isabela. Universidade Federal do Rio de Janeiro; BrasilFil: Koeller, Carolina M.. Universidade Federal do Rio de Janeiro; Brasil. University at Buffalo; Estados UnidosFil: Costa, Tatiana F. R.. Universidade Federal do Rio de Janeiro; BrasilFil: Rodrigues, Nathalia S.. Universidade Federal do Rio de Janeiro; BrasilFil: Lima, Ana P.. Universidade Federal do Rio de Janeiro; BrasilFil: Atella, Georgia C.. Universidade Federal do Rio de Janeiro; BrasilFil: Rocha de Miranda, Kildare. Universidade Federal do Rio de Janeiro; Brasil. Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem; BrasilFil: Schoijet, Alejandra Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: de Alcântara Machado, Ednildo. Universidade Federal do Rio de Janeiro; BrasilFil: Heise, Norton. Universidade Federal do Rio de Janeiro; Brasi

Ancoras glicosilfosfatidilinositol em Trypanosoma cruzi e sua biossintese

BV UNIFESP: Teses e dissertaçõe

The dihydroxyacetonephosphate pathway for biosynthesis of ether lipids in Leishmania mexicana promastigotes

Biosynthetic studies using both [14C]- and [32P]-labelled substrates and a cell-free system to synthesise 1-O-alkyl moieties in glycerolipids, have shown that the three initial steps in ether-lipid biosynthesis in Leishmania mexicana promastigotes resemble those described for mammals and are associated with glycosomes. Purified glycosomes were able to sequentially synthesise the first intermediates of the ether-lipid biosynthetic pathway [acyl-dihydroxyacetonephosphate (DHAP), alkyl-DHAP and acyl/alkyl-glycerol-3-phosphate (G3P)] when incubated in the presence of radiolabelled DHAP, palmitoyl-CoA, hexadecanol and NADPH. However, when glycosomes were incubated under the same conditions in the presence of radiolabelled G3P, a rapid synthesis of acyl-G3P and phosphatidic acid was observed without any formation of alkyl-G3P, suggesting that the enzyme alkyl-synthase recognises only acyl-DHAP as substrate. Both the DHAP acyltransferase (DHAP-AT) and alkyl-DHAP synthase activities were located inside glycosomes whereas the alkyl/acyl-DHAP oxidoreductase activity was associated with the cytoplasmic face of the glycosomal membrane. The G3P acyltransferase (G3P-AT) and lyso-phosphatidic acid acyltransferase activities were not found inside glycosomes. The results suggest that the DHAP-AT and G3P-AT activities are catalysed by two distinct enzymes associated with different sub-cellular compartments

CHARACTERIZATION OF A PHOSPHOLIPASE C-RESISTANT INOSITOL CONTAINING GLYCOLIPID FROM TRYPANOSOMA-CRUZI

Since glycosylphosphatidylinositol is the most common form of attachment of proteins to membranes in T. cruzi, and that this parasite depends on surface-mediated interactions for survival within the vector and mammalian host, it is probable that a drug which interfers with the metabolism of glycosylphosphatidylinositol (GPI) could be successfully employed in chemotherapy. Over the last few years several groups have been characterizing this mode of attachment in T. cruzi and more recently we have been concentrating our efforts on the identification of candidate precursors for protein anchors in metacyclic trypomastigotes. Previously detected GPI heterogeneity regarding solubilization of a major stage-specific antigen (1G7-Ag) by phospholipase C led us to investigate whether biosynthetic precursors with similar properties could also be identified. Two glycolipid species whose migration properties resemble glycolipids A and C of T. brucei were amenable to biosynthetic radiolabelling with palmitic acid, inositol, ethanolamine, glucosamine and mannose. Following purification,these species were submitted to classical GPI diagnostic treatments. In both cases digestion with GPI-specific phospholipase D (GPIPLD) produced phospatidic acid and treatment with either mild base or phospholipase A(2) (PLA(2)) produced free fatty acid, indicating an acylation at least at position 2 of the glycerol. The glycolipid A-like species proved to be susceptible to solubilization by PIPLC of B. thuringiensis and by GPIPLC of T. brucei and the glycolipid C-like material proved to be fully resistant to both lipases. Although the glycolipid A-like species indeed presents these and other properties compatible with a precursor for the chemically characterized 1 G7-Ag anchor, the PLC-resistant species which is completely insensitive to nitrous acid deamination might be an exception to the general finding of a non-acetylated glucosamine in the GPI moieties so far described.JOHNS HOPKINS UNIV,DEPT BIOL CHEM,BALTIMORE,MD 21205Web of Scienc