A Physiological Approach to Explore How Thioredoxin-Glutathione Reductase (TGR) and Peroxiredoxin (Prx) Eliminate H2O2 in Cysticerci of Taenia.

Peroxiredoxins (Prxs) and glutathione peroxidases (GPxs) are the main enzymes of the thiol-dependent antioxidant systems responsible for reducing the H2O2 produced via aerobic metabolism or parasitic organisms by the host organism. These antioxidant systems maintain a proper redox state in cells. The cysticerci of Taenia crassiceps tolerate millimolar concentrations of this oxidant. To understand the role played by Prxs in this cestode, two genes for Prxs, identified in the genome of Taenia solium (TsPrx1 and TsPrx3), were cloned. The sequence of the proteins suggests that both isoforms belong to the class of typical Prxs 2-Cys. In addition, TsPrx3 harbors a mitochondrial localization signal peptide and two motifs (-GGLG- and -YP-) associated with overoxidation. Our kinetic characterization assigns them as thioredoxin peroxidases (TPxs). While TsPrx1 and TsPrx3 exhibit the same catalytic efficiency, thioredoxin-glutathione reductase from T. crassiceps (TcTGR) was five and eight times higher. Additionally, the latter demonstrated a lower affinity (>30-fold) for H2O2 in comparison with TsPrx1 and TsPrx3. The TcTGR contains a Sec residue in its C-terminal, which confers additional peroxidase activity. The aforementioned aspect implies that TsPrx1 and TsPrx3 are catalytically active at low H2O2 concentrations, and the TcTGR acts at high H2O2 concentrations. These results may explain why the T. crassiceps cysticerci can tolerate high H2O2 concentrations.

Evaluating the effect of curcumin on the metacestode of Taenia crassiceps.

Curcumin, a curcuminoid present in the rhizome of the plant Curcuma longa has multiple pharmacological effects including anticarcinogenic and anti-inflammatory properties. This work evaluates the anthelmintic effect of the curcumin molecule (98% pure) on Taenia crassiceps cysticerci viability in vitro. Cysticerci incubated in the presence of increasing concentrations of curcumin showed a dose-dependent mortality correlated with a significant increase in the production of reactive oxygen species and a partial inhibition of thioredoxin-glutathione reductase, the only disulfide reductase present in these parasites. At 500 µM curcumin, a 100% of cysticerci lethality was obtained after 2 h of treatment. These results suggest the curcumin-induced oxidative stress could be in the origin of the anthelminthic effect of curcumin. Mice with cysticerci were injected intraperitoneally with 20, 40, or 60 mM curcumin daily for 30 days. A decrease in the burden of cysticerci (46%) was observed with a 60 mM dose of curcumin, supporting this compound as a potential anthelmintic drug.

Correction: Effect of curcuminoids and curcumin derivate products on thioredoxin-glutathione reductase from Taenia crassiceps cysticerci. Evidence suggesting a curcumin oxidation product as a suitable inhibitor.

[This corrects the article DOI: 10.1371/journal.pone.0220098.].

Effect of curcuminoids and curcumin derivate products on thioredoxin-glutathione reductase from Taenia crassiceps cysticerci. Evidence suggesting a curcumin oxidation product as a suitable inhibitor.

Curcuma is a traditional ingredient of some Eastern cuisines, and the spice is heralded for its antitumoral and antiparasitic properties. In this report, we examine the effect of the curcuminoides which include curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), as well as curcumin degradation products on thioredoxin glutathione reductase from Taenia crassiceps cysticerci Results revealed that both DMC and BDMC were inhibitors of TGR activity in the micromolar concentration range. By contrast, the inhibitory ability of curcumin was a time-dependent process. Kinetic and spectroscopical evidence suggests that an intermediary compound of curcumin oxidation, probably spiroepoxide, is responsible. Preincubation of curcumin in the presence of NADPH, but not glutathione disulfide (GSSG), resulted in the loss of its inhibitory ability, suggesting a reductive stabilizing effect. Similarly, preincubation of curcumin with sulfhydryl compounds fully protected the enzyme from inhibition. Degradation products were tested for their inhibitory potential, and 4-vinylguaiacol was the best inhibitor (IC50 = 12.9 µM), followed by feruloylmethane (IC50 = 122 µM), vanillin (IC50 = 127 µM), and ferulic aldehyde (IC50 = 180 µM). The acid derivatives ferulic acid (IC50 = 465 µM) and vanillic acid (IC50 = 657 µM) were poor inhibitors. On the other hand, results from docking analysis revealed a common binding site on the enzyme for all the compounds, albeit interacting with different amino acid residues. Dissociation constants obtained from the docking were in accord with the inhibitory efficiency of the curcumin degradation products.

A reduction in ecological niche for Trypanosoma cruzi-infected triatomine bugs.

BACKGROUND: Theory predicts that parasites can affect and thus drive their hosts' niche. Testing this prediction is key, especially for vector-borne diseases including Chagas disease. Here, we examined the niche use of seven triatomine species that occur in Mexico, based on whether they are infected or not with Trypanosoma cruzi, the vectors and causative parasites of Chagas disease, respectively. Presence data for seven species of triatomines (Triatoma barberi, T. dimidiata, T. longipennis, T. mazzottii, T. pallidipennis, T. phyllosoma and T. picturata) were used and divided into populations infected and not infected by T. cruzi. Species distribution models were generated with Maxent 3.3.3k. Using distribution models, niche analysis tests of amplitude and distance to centroids were carried out for infected vs non-infected populations within species. RESULTS: Infected populations of bugs of six out of the seven triatomine species showed a reduced ecological space compared to non-infected populations. In all but one case (T. pallidipennis), the niche used by infected populations was close to the niche centroid of its insect host. CONCLUSIONS: Trypanosoma cruzi may have selected for a restricted niche amplitude in triatomines, although we are unaware of the underlying reasons. Possibly the fact that T. cruzi infection bears a fitness cost for triatomines is what narrows the niche breadth of the insects. Our results imply that Chagas control programmes should consider whether bugs are infected in models of triatomine distribution.

Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala).

A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), and a putative thioredoxin reductase (TrxR) was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.

The Architecture of Thiol Antioxidant Systems among Invertebrate Parasites.

The use of oxygen as the final electron acceptor in aerobic organisms results in an improvement in the energy metabolism. However, as a byproduct of the aerobic metabolism, reactive oxygen species are produced, leaving to the potential risk of an oxidative stress. To contend with such harmful compounds, living organisms have evolved antioxidant strategies. In this sense, the thiol-dependent antioxidant defense systems play a central role. In all cases, cysteine constitutes the major building block on which such systems are constructed, being present in redox substrates such as glutathione, thioredoxin, and trypanothione, as well as at the catalytic site of a variety of reductases and peroxidases. In some cases, the related selenocysteine was incorporated at selected proteins. In invertebrate parasites, antioxidant systems have evolved in a diversity of both substrates and enzymes, representing a potential area in the design of anti-parasite strategies. The present review focus on the organization of the thiol-based antioxidant systems in invertebrate parasites. Differences between these taxa and its final mammal host is stressed. An understanding of the antioxidant defense mechanisms in this kind of parasites, as well as their interactions with the specific host is crucial in the design of drugs targeting these organisms.

Understanding transmissibility patterns of Chagas disease through complex vector-host networks.

Chagas disease is one of the most important vector-borne zoonotic diseases in Latin America. Control strategies could be improved if transmissibility patterns of its aetiologic agent, Trypanosoma cruzi, were better understood. To understand transmissibility patterns of Chagas disease in Mexico, we inferred potential vectors and hosts of T. cruzi from geographic distributions of nine species of Triatominae and 396 wild mammal species, respectively. The most probable vectors and hosts of T. cruzi were represented in a Complex Inference Network, from which we formulated a predictive model and several associated hypotheses about the ecological epidemiology of Chagas disease. We compiled a list of confirmed mammal hosts to test our hypotheses. Our tests allowed us to predict the most important potential hosts of T. cruzi and to validate the model showing that the confirmed hosts were those predicted to be the most important hosts. We were also able to predict differences in the transmissibility of T. cruzi among triatomine species from spatial data. We hope our findings help drive efforts for future experimental studies.

Proteolytic activity of extracellular products from Arthrobotrys musiformis and their effect in vitro against Haemonchus contortus infective larvae.

Arthrobotrys musiformis is a nematophagous fungus with potential for the biological control of Haemonchus contortus larvae. This study aimed to identify and demonstrate the proteolytic activity of extracellular products from A musiformis cultured in a liquid medium against H contortus infective larvae. A musiformis was cultured on a solid medium and further grown in a liquid medium, which was then processed through ion exchange and hydrophobic interaction chromatography. The proteolytic activity of the purified fraction was assayed with either gelatin or bovine serum albumin as substrate. Optimum proteolytic activity was observed at pH 8 and a temperature of 37°C. Results obtained with specific inhibitors suggest the enzyme belongs to the serine-dependent protease family. The purified fraction concentrate from A musiformis was tested against H contortus infective larvae. A time-dependent effect was observed with 77 per cent immobility after 48 hours incubation, with alteration of the sheath. It is concluded that A musiformis is a potential candidate for biological control because of its resistant structures and also because of its excretion of extracellular products such as proteases. The present study contributes to the identification of one of the in vitro mechanisms of action of A musiformis, namely the extracellular production of proteases against H contortus infective larvae. More investigations should be undertaken into how these products could be used to decrease the nematode population in sheep flocks under field conditions, thereby improving animal health while simultaneously diminishing the human and environmental impact of chemical-based drugs.

Membrane potential regulates mitochondrial ATP-diphosphohydrolase activity but is not involved in progesterone biosynthesis in human syncytiotrophoblast cells.

ATP-diphosphohydrolase is associated with human syncytiotrophoblast mitochondria. The activity of this enzyme is implicated in the stimulation of oxygen uptake and progesterone synthesis. We reported previously that: (1) the detergent-solubilized ATP-diphosphohydrolase has low substrate specificity, and (2) purine and pyrimidine nucleosides, tri- or diphosphates, are fully dephosphorylated in the presence of calcium or magnesium (Flores-Herrera 1999, 2002). In this study we show that ATP-diphosphohydrolase hydrolyzes first the nucleoside triphosphate to nucleoside diphosphate, and then to nucleotide monophosphate, in the case of all tested nucleotides. The activation energies (Ea) for ATP, GTP, UTP, and CTP were 6.06, 4.10, 6.25, and 5.26 kcal/mol, respectively; for ADP, GDP, UDP, and CDP, they were 4.67, 5.42, 5.43, and 6.22 kcal/mol, respectively. The corresponding Arrhenius plots indicated a single rate-limiting step for each hydrolyzed nucleoside, either tri- or diphosphate. In intact mitochondria, the ADP produced by ATP-diphosphohydrolase activity depolarized the membrane potential (ΔΨm) and stimulated oxygen uptake. Mitochondrial respiration showed the state-3/state-4 transition when ATP was added, suggesting that ATP-diphosphohydrolase and the F1F0-ATP synthase work in conjunction to avoid a futile cycle. Substrate selectivity of the ATP-diphosphohydrolase was modified by ΔΨm (i.e. ATP was preferred over GTP when the inner mitochondrial membrane was energized). In contrast, dissipation of ΔΨm by CCCP produced a loss of substrate specificity and so the ATP-diphosphohydrolase was able to hydrolyze ATP and GTP at the same rate. In intact mitochondria, ATP hydrolysis increased progesterone synthesis as compared with GTP. Although dissipation of ΔΨm by CCCP decreased progesterone synthesis, NADPH production restores steroidogenesis. Overall, our results suggest a novel physiological role for ΔΨm in steroidogenesis.

The alternative NADH dehydrogenase is present in mitochondria of some animal taxa.

The distribution of the alternative NADH dehydrogenase (NDH-2) in the living world was explored. The enzyme, although present in representatives of all living kingdoms, does not have a universal distribution. With the exception of ε-proteobacteria, the enzyme was found in all eubacterial groups. In contrast with the known presence of the NDH-2 in Archaea, the alternative oxidase (AOX) is absent in this group. With regard to the Eukarya domain, the NDH-2 was found in representatives of Protista, Fungi, Plantae, and Animalia. In the latter, however, the presence of the enzyme was restricted to some primitive Metazoa (Placozoa and Cnidaria), and two members of the Deuterostomate lineage of the Bilateria (Echinodermata and Urochordata). No evidence for the presence of the NDH-2 was found in any representative of the Protostomate branch of the Bilateria, contrasting with the existence of the AOX in this same group. It is worth mentioning that those animal species containing the NDH-2 also have an AOX. The actual distribution of the NDH-2 in the various living kingdoms is discussed within the framework of the endosymbiotic theory; in addition, a hypothesis is proposed to explain the disappearance of the alternative NDH-2 and AOX from the majority of the animals.

Trypanocidal activity of the ethyl esters of N-propyl and N-isopropyl oxamates on intracellular amastigotes of Trypanosoma cruzi acute infected mice.

In this investigation we studied the trypanocidal activity of the ethyl esters of N-propyl (Et-NPOX) and N-isopropyl (Et-NIPOX) oxamates on bloodstream trypomastigotes and on the clinically relevant intracellular amastigotes of Trypanosoma cruzi acute infected mice. In the infected and treated mice, the levels of parasitemia were drastically reduced between days 15 and 20 of treatment and almost to zero between days 35 and 40. We also found that Et-NPOX completely eliminated amastigote nests in the myocardium of mice infected with INC-5 or NINOA T. cruzi strain, and in skeletal muscle the reduction in the number of amastigote nests was between 60 and 80% in both strains. Also, Et-NIPOX reduced by 60-80% the number of amastigote nests in the myocardium and skeletal muscle of mice infected with these T. cruzi strains. In contrast, nifurtimox, used for comparison, produced a reduction of amastigote nests of only 20-40% in the studied tissues in both strains.

Signaling the signal, cyclic AMP-dependent protein kinase inhibition by insulin-formed H2O2 and reactivation by thioredoxin.

Catecholamines in adipose tissue promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. Here we show that H(2)O(2) generated by insulin in rat adipocytes impaired cAMP-mediated amplification cascade of lipolysis. These micromolar concentrations of H(2)O(2) added before cAMP suppressed cAMP activation of type IIbeta cyclic AMP-dependent protein kinase (PKA) holoenzyme, prevented hormone-sensitive lipase translocation from cytosol to storage droplets, and inhibited lipolysis. Similarly, H(2)O(2) impaired activation of type IIalpha PKA holoenzyme from bovine heart and from that reconstituted with regulatory IIalpha and catalytic alpha subunits. H(2)O(2) was ineffective (a) if these PKA holoenzymes were preincubated with cAMP, (b) if added to the catalytic alpha subunit, which is active independently of cAMP activation, and (c) if the catalytic alpha subunit was substituted by its C199A mutant in the reconstituted holoenzyme. H(2)O(2) inhibition of PKA activation remained after H(2)O(2) elimination by gel filtration but was reverted with dithiothreitol or with thioredoxin reductase plus thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H(2)O(2) incubation. These data strongly suggest that H(2)O(2) promotes the formation of an intersubunit disulfide bond, impairing cAMP-dependent PKA activation. Phylogenetic analysis showed that Cys-97 is conserved only in type II regulatory subunits and not in type I regulatory subunits; hence, the redox regulation mechanism described is restricted to type II PKA-expressing tissues. In conclusion, phylogenetic analysis results, selective chemical behavior, and the privileged position in holoenzyme lead us to suggest that Cys-97 in regulatory IIalpha or IIbeta subunits is the residue forming the disulfide bond with Cys-199 in the PKA catalytic alpha subunit. A new molecular point for cross-talk among heterologous signal transduction pathways is demonstrated.

The bacterial-like lactate shuttle components from heterotrophic Euglena gracilis.

The structural and kinetic analyses of the components of the lactate shuttle from heterotrophic Euglena gracilis were carried out. Mitochondrial membrane-bound, NAD(+)-independent d-lactate dehydrogenase (d-iLDH) was purified by solubilization with CHAPS and heat treatment. The active enzyme was a 62-kDa monomer containing non-covalently bound FAD as cofactor. d-iLDH was specific for d-lactate and it was able to reduce quinones of different redox potential values. Oxalate and l-lactate were mixed-type inhibitors of d-iLDH. Mitochondrial l-iLDH also catalyzed the reduction of quinones, but it was inactivated during the extraction with detergents. Both l-iLDH and d-iLDH were inhibited by the specific flavoprotein-inhibitor diphenyleneiodonium, suggesting that l-iLDH was also a flavoprotein. Affinity chromatography revealed that the E. gracilis cytosolic fraction contained two types of NAD(+)-dependent LDH specific for the generation of d- and l-lactate (d-nLDH and l-nLDH, respectively). These two enzymes were tetramers of 126-132 kDa and showed an ordered bi-bi kinetic mechanism. Kinetic properties were different in both enzymes. Pyruvate reduction by d-nLDH was inhibited by its two products; the d-lactate oxidation was 40-fold lower than forward reaction. l-lactate oxidation by l-nLDH was not detected, whereas pyruvate reduction was activated by fructose-1, 6-bisphosphate, K(+) or NH(4)(+). Interestingly, membrane-bound l- and d-lactate dehydrogenases with quinone reductase activity have been only detected in bacteria, whereas the activity of soluble d-nLDH has been identified in bacteria and some yeast. Also, FBP-activated l-nLDH has been found solely in lactic bacteria. Based on their similar kinetic and structural characteristics, a possible common origin among bacterial and E. gracilis lactic dehydrogenase enzymes is discussed.