Analysis of the effect of methyl 2-acetamide-3-methylquinoxaline-7-carboxylate 1,4-di-N-oxide on the relative expression of the trypanothione reductase gene in Trypanosoma cruzi epimastigotes.

In recent decades, some quinoxaline 1,4-di-N-oxide derivatives have been shown to have better trypanocidal activity than the reference drugs; however, their mechanism of action is not yet clear, although it is suggested that they mainly produce reactive oxygen species that cause oxidative stress and parasite death. Trypanosoma cruzi relies on the enzyme trypanothione reductase, among others, to defend itself against oxidative stress. With the aim of contributing to the elucidation of the mechanism of action of quinoxaline 1,4-di-N-oxide derivatives on Trypanosoma cruzi, this study was carried out to evaluate the effect of methyl 2-amide-3-methylquinoxaline-7-carboxylate 1,4-di-N-oxide (compound M-8) on the expression of the trypanothione reductase gene in an in vitro model on Trypanosoma cruzi epimastigotes of the CL-Brener strain. The results show that compound M-8 does not cause a significant effect on the trypanothione reductase gene, suggesting a mechanism of action not related to oxidative stress.

Gamma-glutamylcysteine synthetase and tryparedoxin 1 exert high control on the antioxidant system in Trypanosoma cruzi contributing to drug resistance and infectivity.

Trypanothione (T(SH)2) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH)2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH)2 synthesis by 60-74% and 15-31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH)2 concentration, whereas TryS overexpression did not render an increase in T(SH)2 levels as a consequence of high T(SH)2 degradation. The peroxide reduction flux was controlled for 64-73% by TXN1, 17-20% by TXNPx and 11-16% by TryR. TXN1 and TryR overexpression increased H2O2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH)2 antioxidant pathway may compromise the parasite's viability and infectivity.

Use of the ptxD gene as a portable selectable marker for chloroplast transformation in Chlamydomonas reinhardtii.

Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.

DNA-based identification reveals illegal trade of threatened shark species in a global elasmobranch conservation hotspot.

Here, we report trading of endangered shark species in a world hotspot for elasmobranch conservation in Brazil. Data on shark fisheries are scarce in Brazil, although the northern and northeastern regions have the highest indices of shark bycatch. Harvest is made primarily with processed carcasses lacking head and fins, which hampers reliable species identification and law enforcement on illegal catches. We used partial sequences of two mitochondrial genes (COI and/or NADH2) to identify 17 shark species from 427 samples being harvested and marketed on the northern coast of Brazil. Nine species (53%) are listed under some extinction threat category according to Brazilian law and international authorities (IUCN - International Union for Conservation of Nature; CITES - Convention on International Trade of Endangered Species of Wild Fauna and Flora). The number increases to 13 (76%) if we also consider the Near Threatened category. Hammerhead sharks are under threat worldwide, and composed 18.7% of samples, with Sphyrna mokarran being the fourth most common species among samples. As illegal trade of threatened shark species is a worldwide conservation problem, molecular identification of processed meat or specimens lacking diagnostic body parts is a highly effective tool for species identification and law enforcement.

Molecular species identification of Trichuris trichiura in African green monkey on St. Kitts, West Indies.

The population of African green monkeys (AGM, Chlorocebus aethiops sabaeus) on St. Kitts, West Indies is believed to be as large as or greater than the human population. Interactions with humans are frequent and the pathogens carried by AGM, such as Trichuris spp., may pose a risk to humans. The objectives of this study were to assess the use of molecular methods for diagnosing Trichuris spp. in AGM and compare its DNA sequences to those of Trichuris spp. found in other non-human primates and humans. Fecal samples were collected from trapped and individually housed AGM between January and December 2015 and analysed using fecal flotation with Sheather's sugar flotation solution and PCR amplification and DNA sequencing of 18S rRNA and ITS2 fragments. Phylogenetic analysis was performed. 91% (81/89) and 55.4% (31/56) were Trichuris spp. positive by fecal flotation and PCR, respectively. Both AGM-NADH1 gene and T. trichiura-18S rRNA gene showed no variations in sequence and were 100% identical to corresponding sequences deposited in GenBank. Nevertheless Trichuris ITS2 showed some diversities among 12 sequences, which was <5%. Phylogenetic analysis of ITS2 put Trichuris spp. in Kittitian AGM into the same clades of T. trichiura found in human and other non-human primates in many other geographical regions. These data confirm that AGM are reservoirs for T. trichiura in humans. We suggest a one health approach to curtail enteric parasitic infections in human populations in the insular country.

Differential oxidative stress tolerance of Streptococcus mutans isolates affects competition in an ecological mixed-species biofilm model.

Streptococcus mutans strongly influences the development of pathogenic biofilms associated with dental caries. Our understanding of S. mutans behaviour in biofilms is based on a few well-characterized laboratory strains; however, individual isolates vary widely in genome content and virulence-associated phenotypes, such as biofilm formation and environmental stress sensitivity. Using an ecological biofilm model, we assessed the impact of co-cultivation of several S. mutans isolates with Streptococcus oralis and Actinomyces naeslundii on biofilm composition following exposure to sucrose. The laboratory reference strain S. mutans UA159 and clinical isolates Smu44 (most aciduric), Smu56 (altered biofilm formation) and Smu81 (more sensitive to oxidative stress) were used. Our data revealed S. mutans isolates varied in their ability to compete and become dominant in the biofilm after the addition of sucrose, and this difference correlated with sensitivity to H2 O2 produced by S. oralis. Smu81 was particularly sensitive to H2 O2 and could not compete with S. oralis in mixed-species biofilm, despite forming robust biofilms on its own. Thus, diminished oxidative stress tolerance in S. mutans isolates can impair their ability to compete in complex biofilms, even in the presence of sucrose, which could influence the progression of a healthy biofilm community to one capable of causing disease.

Aluminum exposure at human dietary levels promotes vascular dysfunction and increases blood pressure in rats: A concerted action of NAD(P)H oxidase and COX-2.

Aluminum (Al) is a non-essential metal and a significant environmental contaminant and is associated with a number of human diseases including cardiovascular disease. We investigated the effects of Al exposure at doses similar to human dietary levels on the cardiovascular system over a 60day period. Wistar male rats were divided into two major groups and received orally: 1) Low aluminum level - rats were subdivided and treated for 60days as follows: a) Untreated - ultrapure water; b) AlCl3 at a dose of 8.3mg/kg bw for 60days, representing human Al exposure by diet; and 2) High aluminum level - rats were subdivided and treated for 42days as follows: C) Untreated - ultrapure water; d) AlCl3 at 100mg/kg bw for 42days, representing a high level of human exposure to Al. Effects on systolic blood pressure (SBP) and vascular function of aortic and mesenteric resistance arteries (MRA) were studied. Endothelium and smooth muscle integrity were evaluated by concentration-response curves to acetylcholine (ACh) and sodium nitroprusside. Vasoconstrictor responses to phenylephrine (Phe) in the presence and absence of endothelium and in the presence of the NOS inhibitor L-NAME, the potassium channels blocker TEA, the NAD(P)H oxidase inhibitor apocynin, superoxide dismutase (SOD), the non-selective COX inhibitor indomethacin and the selective COX-2 inhibitor NS 398 were analyzed. Vascular reactive oxygen species (ROS), lipid peroxidation and total antioxidant capacity, were measured. The mRNA expressions of eNOS, NAD(P)H oxidase 1 and 2, SOD1, COX-2 and thromboxane A2 receptor (TXA-2 R) were also investigated. Al exposure at human dietary levels impaired the cardiovascular system and these effects were almost the same as Al exposure at much higher levels. Al increased SBP, decreased ACh-induced relaxation, increased response to Phe, decreased endothelial modulation of vasoconstrictor responses, the bioavailability of nitric oxide (NO), the involvement of potassium channels on vascular responses, as well as increased ROS production from NAD(P)H oxidase and contractile prostanoids mainly from COX-2 in both aorta and mesenteric arteries. Al exposure increased vascular ROS production and lipid peroxidation as well as altered the antioxidant status in aorta and MRA. Al decreased vascular eNOS and SOD1 mRNA levels and increased the NAD(P)H oxidase 1, COX-2 and TXA-2 R mRNA levels. Our results point to an excess of ROS mainly from NAD(P)H oxidase after Al exposure and the increased vascular prostanoids from COX-2 acting in concert to decrease NO bioavailability, thus inducing vascular dysfunction and increasing blood pressure. Therefore, 60-day chronic exposure to Al, which reflects common human dietary Al intake, appears to pose a risk for the cardiovascular system.

The Enzymatic and Structural Basis for Inhibition of Echinococcus granulosus Thioredoxin Glutathione Reductase by Gold(I).

AIMS: New drugs are needed to treat flatworm infections that cause severe human diseases such as schistosomiasis. The unique flatworm enzyme thioredoxin glutathione reductase (TGR), structurally different from the human enzyme, is a key drug target. Structural studies of the flatworm Echinococcus granulosus TGR, free and complexed with AuI-MPO, a novel gold inhibitor, together with inhibition assays were performed. RESULTS: AuI-MPO is a potent TGR inhibitor that achieves 75% inhibition at a 1:1 TGR:Au ratio and efficiently kills E. granulosus in vitro. The structures revealed salient insights: (i) unique monomer-monomer interactions, (ii) distinct binding sites for thioredoxin and the glutaredoxin (Grx) domain, (iii) a single glutathione disulfide reduction site in the Grx domain, (iv) rotation of the Grx domain toward the Sec-containing redox active site, and (v) a single gold atom bound to Cys519 and Cys573 in the AuI-TGR complex. Structural modeling suggests that these residues are involved in the stabilization of the Sec-containing C-terminus. Consistently, Cys→Ser mutations in these residues decreased TGR activities. Mass spectroscopy confirmed these cysteines are the primary binding site. INNOVATION: The identification of a primary site for gold binding and the structural model provide a basis for gold compound optimization through scaffold adjustments. CONCLUSIONS: The structural study revealed that TGR functions are achieved not only through a mobile Sec-containing redox center but also by rotation of the Grx domain and distinct binding sites for Grx domain and thioredoxin. The conserved Cys519 and Cys573 residues targeted by gold assist catalysis through stabilization of the Sec-containing redox center. Antioxid. Redox Signal. 27, 1491-1504.

Cloning, Expression and Characterization of Recombinant, NADH Oxidase from Giardia lamblia.

The NADH oxidase family of enzymes catalyzes the oxidation of NADH by reducing molecular O2 to H2O2, H2O or both. In the protozoan parasite Giardia lamblia, the NADH oxidase enzyme (GlNOX) produces H2O as end product without production of H2O2. GlNOX has been implicated in the parasite metabolism, the intracellular redox regulation and the resistance to drugs currently used against giardiasis; therefore, it is an interesting protein from diverse perspectives. In this work, the GlNOX gene was amplified from genomic G. lamblia DNA and expressed in Escherichia coli as a His-Tagged protein; then, the enzyme was purified by immobilized metal affinity chromatography, characterized, and its properties compared with those of the endogenous enzyme previously isolated from trophozoites (Brown et al. in Eur J Biochem 241(1):155-161, 1996). In comparison with the trophozoite-extracted enzyme, which was scarce and unstable, the recombinant heterologous expression system and one-step purification method produce a stable protein preparation with high yield and purity. The recombinant enzyme mostly resembles the endogenous protein; where differences were found, these were attributable to methodological discrepancies or artifacts. This homogenous, pure and functional protein preparation can be used for detailed structural or functional studies of GlNOX, which will provide a deeper understanding of the biology and pathogeny of G. lamblia.

Alpha1beta1 and integrin-linked kinase interact and modulate angiotensin II effects in vascular smooth muscle cells.

The effects of angiotensin II (Ang II) on vascular smooth muscle cells (VSMC) are modulated by reactive oxygen species (ROS) and also involve integrin engagement. However, the potential link between alpha1beta1 integrin signaling with NOX system and their combined contribution to Ang II effects on VSMC have not been investigated. We aimed to elucidate the moslecular mechanisms underlying the activation of these two pathways in Ang II effects on VSMC. Ang II-induced VSMC migration (2-fold increase) and proliferation (2.5-fold increase) is modulated by alpha1beta1 integrin, being inhibited by obtustatin, a specific alpha1beta1 integrin blocker. Ang II also stimulates ROS production in VSMC (140%) that is NOX1 dependent, being completely inhibited in NOX1 silenced cells. The ROS production develops in two peaks, and the second peak is maintained by NOX2 activation. Apocynin and obtustatin inhibit the NOX2-associated second peak, but not the first peak of ROS production, which is related to NOX1 activation. Corroborating the involvement of alpha1beta1 integrin, the pretreatment of VSMC with obtustatin impaired Ang II-induced FAK phosphorylation, AKT activation, p21 degradation and the increase of ILK expression. Silencing of ILK blocked cell migration, AKT phosphorylation and the second peak of ROS, but partially inhibits (70%) VSMC proliferation induced by Ang II. The data demonstrate a novel role for NOX2 in Ang II effects on VSMC, and suggest alpha1beta1 integrin and ILK as target molecules to the development of more effective therapeutic interventions in cardiovascular diseases.

New enzymatic pathways for the reduction of reactive oxygen species in Entamoeba histolytica.

BACKGROUND: Entamoeba histolytica, an intestinal parasite that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. A flavodiiron protein and a rubrerythrin have been characterized in this human pathogen, although their physiological reductants have not been identified. METHODS: The present work deals with biochemical studies performed to reach a better understanding of the kinetic and structural properties of rubredoxin reductase and two ferredoxins from E. histolytica. RESULTS: We complemented the characterization of two different metabolic pathways for O2 and H2O2 detoxification in E. histolytica. We characterized a novel amoebic protein with rubredoxin reductase activity that is able to catalyze the NAD(P)H-dependent reduction of heterologous rubredoxins, amoebic rubrerythrin and flavodiiron protein but not ferredoxins. In addition, the protein exhibited an NAD(P)H oxidase activity, which generates hydrogen peroxide from molecular oxygen. We describe how different ferredoxins were also efficient reducing substrates for both flavodiiron protein and rubrerythrin. CONCLUSIONS: The enzymatic systems herein characterized could contribute to the in vivo detoxification of O2 and H2O2, playing a key role for the parasite defense against reactive oxidant species. GENERAL SIGNIFICANCE: To the best of our knowledge this is the first characterization of a eukaryotic rubredoxin reductase, including a novel kinetic study on ferredoxin-dependent reduction of flavodiiron and rubrerythrin proteins.

Microglial cells are involved in the susceptibility of NADPH oxidase knockout mice to 6-hydroxy-dopamine-induced neurodegeneration.

We explored the impact of Nox-2 in modulating inflammatory-mediated microglial responses in the 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD) model. Nox1 and Nox2 gene expression were found to increase in striatum, whereas a marked increase of Nox2 expression was observed in substantia nigra (SN) of wild-type (wt) mice after PD induction. Gp91(phox-/-) 6-OHDA-lesioned mice exhibited a significant reduction in the apomorphine-induced rotational behavior, when compared to wt mice. Immunolabeling assays indicated that striatal 6-OHDA injections reduced the number of dopaminergic (DA) neurons in the SN of wt mice. In gp91(phox-/-) 6-OHDA-lesioned mice the DA degeneration was negligible, suggesting an involvement of Nox in 6-OHDA-mediated SN degeneration. Gp91(phox-/-) 6-OHDA-lesioned mice treated with minocycline, a tetracycline derivative that exerts multiple anti-inflammatory effects, including microglial inhibition, exhibited increased apomorphine-induced rotational behavior and degeneration of DA neurons after 6-OHDA injections. The same treatment also increased TNF-α release and potentiated NF-κB activation in the SN of gp91(phox-/-)-lesioned mice. Our results demonstrate for the first time that inhibition of microglial cells increases the susceptibility of gp91(phox-/-) 6-OHDA lesioned mice to develop PD. Blockade of microglia leads to NF-κB activation and TNF-α release into the SN of gp91(phox-/-) 6-OHDA lesioned mice, a likely mechanism whereby gp91(phox-/-) 6-OHDA lesioned mice may be more susceptible to develop PD after microglial cell inhibition. Nox2 adds an essential level of regulation to signaling pathways underlying the inflammatory response after PD induction.

Heme modulates intestinal epithelial cell activation: involvement of NADPHox-derived ROS signaling.

In many gut chronic inflammatory conditions, intestinal epithelium (IE) is deprived of the protection of the mucus secreted by IE-specialized cells. In these events, bleeding and subsequent lysis of erythrocytes are common. This may lead to the release of high amounts of heme in the intestinal lumen, which interacts with IE. Previous works from our group have shown that heme itself is a proinflammatory molecule, activating a number of phlogistic signaling events in a nicotinamide adenine dinucleotide phosphate oxidase (NADPHox)-dependent manner. In this study, we aim to evaluate the effects of heme upon a well-established nontransformed small intestine epithelial cell lineage (IEC 6). Our results show that free heme evokes intracellular reactive oxygen species (ROS) production by IEC 6 cells, which is inhibited both by pharmacological inhibition with diphenyleneiodonium (10 µM), a NADPHox inhibitor, and small interfering RNA-mediated suppression of NOX1, a constitutive NADPHox isoform present in intestinal epithelial cells. Focal adhesion kinase phosphorylation and actin cytoskeleton polymerization are also induced by heme in a NADPHox-dependent manner. Heme increases monolayer permeability and redistributes key modulators of cell-cell adhesion as zona occludens-1 and E-cadherin proteins via NADPHox signaling. Heme promotes IEC 6 cell migration and proliferation, phenomena also regulated by NADPHox-derived ROS. Heme, in NADPHox-activating concentrations, is able to induce mRNA expression of IL-6, a cytokine implicated in inflammatory and tumorigenic responses. These data indicate a prominent role for heme-derived signaling in the pathophysiology of intestinal mucosa dysfunction and address an important role of NADPHox activity on the pathogenesis of intestinal inflammatory conditions.

Testosterone induces vascular smooth muscle cell migration by NADPH oxidase and c-Src-dependent pathways.

Testosterone has been implicated in vascular remodeling associated with hypertension. Molecular mechanisms underlying this are elusive, but oxidative stress may be important. We hypothesized that testosterone stimulates generation of reactive oxygen species (ROS) and migration of vascular smooth muscle cells (VSMCs), with enhanced effects in cells from spontaneously hypertensive rats (SHRs). The mechanisms (genomic and nongenomic) whereby testosterone induces ROS generation and the role of c-Src, a regulator of redox-sensitive migration, were determined. VSMCs from male Wistar-Kyoto rats and SHRs were stimulated with testosterone (10(-7) mol/L, 0-120 minutes). Testosterone increased ROS generation, assessed by dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence (30 minutes [SHR] and 60 minutes [both strains]). Flutamide (androgen receptor antagonist) and actinomycin D (gene transcription inhibitor) diminished ROS production (60 minutes). Testosterone increased Nox1 and Nox4 mRNA levels and p47phox protein expression, determined by real-time PCR and immunoblotting, respectively. Flutamide, actinomycin D, and cycloheximide (protein synthesis inhibitor) diminished testosterone effects on p47phox. c-Src phosphorylation was observed at 30 minutes (SHR) and 120 minutes (Wistar-Kyoto rat). Testosterone-induced ROS generation was repressed by 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine (c-Src inhibitor) in SHRs and reduced by apocynin (antioxidant/NADPH oxidase inhibitor) in both strains. Testosterone stimulated VSMCs migration, assessed by the wound healing technique, with greater effects in SHRs. Flutamide, apocynin, and 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine blocked testosterone-induced VSMCs migration in both strains. Our study demonstrates that testosterone induces VSMCs migration via NADPH oxidase-derived ROS and c-Src-dependent pathways by genomic and nongenomic mechanisms, which are differentially regulated in VSMCs from Wistar-Kyoto rats and SHRs.

Deep mitochondrial divergence in Baja California populations of an aquilopelagic elasmobranch: the golden cownose ray.

Assessing the realized effect of dispersal in the genetic makeup of a species has significant evolutionary, ecological, and economical consequences. Here, we investigate the genetic diversity and population differentiation in the aquilopelagic golden cownose ray Rhinoptera steindachneri from the Gulf of California (GC) and the Pacific coast of Baja California (PCBC) using the mitochondrial NADH2 gene. Low levels of genetic diversity were found with only 4 polymerase chain reaction-restriction fragment length polymorphism haplotypes among 76 specimens. Pacific coast organisms were fixed for a unique haplotype not shared with rays from the gulf; 92% of GC rays possessed a single NADH2 haplotype not found in the Pacific. This produced significant differentiation between the GC and the PCBC (Φ(CT) = 0.972, P < 0.001). A pronounced phylogeographic pattern was found in which GC haplotypes were reciprocally monophyletic relative to a very divergent Pacific lineage (d = 10%). Our results indicate that despite high dispersal potential, GC and PCBC golden cownose ray populations are characterized by highly divergent mitochondrial lineages. Although more evidence is needed to corroborate the genetic isolation and systematic status of PCBC and GC golden cownose rays, our results suggest a possible cryptic species in the region.

Comparison of gene expression patterns among Leishmania braziliensis clinical isolates showing a different in vitro susceptibility to pentavalent antimony.

INTRODUCTION: Evaluation of Leishmania drug susceptibility depends on in vitro Sb(V) susceptibility assays, which are labour-intensive and may give a biased view of the true parasite resistance. Molecular markers are urgently needed to improve and simplify the monitoring of Sb(V)-resistance. We analysed here the gene expression profile of 21 L. braziliensis clinical isolates in vitro defined as Sb(V)-resistant and -sensitive, in order to identify potential resistance markers. METHODS: The differential expression of 13 genes involved in Sb(V) metabolism, oxidative stress or housekeeping functions was analysed during in vitro promastigote growth. RESULTS: Expression profiles were up-regulated for 5 genes only, each time affecting a different set of isolates (mosaic picture of gene expression). Two genes, ODC (ornithine decarboxylase) and TRYR (trypanothione reductase), showed a significantly higher expression rate in the group of Sb(V)-resistant compared to the group of Sb(V)-sensitive parasites (P<0.01). However, analysis of individual isolates showed both markers to explain only partially the drug resistance. DISCUSSION: Our results might be explained by (i) the occurrence of a pleiotropic molecular mechanism leading to the in vitro Sb(V) resistance and/or (ii) the existence of different epi-phenotypes not revealed by the in vitro Sb(V) susceptibility assays, but interfering with the gene expression patterns.

NADPH oxidases NOX-1 and NOX-2 require the regulatory subunit NOR-1 to control cell differentiation and growth in Neurospora crassa.

We have proposed that reactive oxygen species (ROS) play essential roles in cell differentiation. Enzymes belonging to the NADPH oxidase (NOX) family produce superoxide in a regulated manner. We have identified three distinct NOX subfamilies in the fungal kingdom and have shown that NoxA is required for sexual cell differentiation in Aspergillus nidulans. Here we show that Neurospora crassa NOX-1 elimination results in complete female sterility, decreased asexual development, and reduction of hyphal growth. The lack of NOX-2 did not affect any of these processes but led instead to the production of sexual spores that failed to germinate, even in the presence of exogenous oxidants. The elimination of NOR-1, an ortholog of the mammalian Nox2 regulatory subunit gp67(phox), also caused female sterility, the production of unviable sexual spores, and a decrease in asexual development and hyphal growth. These results indicate that NOR-1 is required for NOX-1 and NOX-2 functions at different developmental stages and establish a link between NOX-generated ROS and the regulation of growth. Indeed, NOX-1 was required for the increased asexual sporulation previously observed in mutants without catalase CAT-3. We also analyzed the function of the penta-EF calcium-binding domain protein PEF-1 in N. crassa. Deletion of pef-1 resulted in increased conidiation but, in contrast to what occurs in Dictyostelium discoideum, the mutation of this peflin did not suppress the phenotypes caused by the lack of NOX-1. Our results support the role of ROS as critical cell differentiation signals and highlight a novel role for ROS in regulation of fungal growth.

Cloning and expression of trypanothione reductase from a New World Leishmania species.

Trypanothione disulfide (T[S]2), an unusual form of glutathione found in parasitic protozoa, plays a crucial role in the regulation of the intracellular thiol redox balance and in the defense against oxidative stress. Trypanothione reductase (TR) is central to the thiol metabolism in all trypanosomatids, including the human pathogens Trypanosoma cruzi, Trypanosoma brucei and Leishmania. Here we report the cloning, sequencing and expression of the TR encoding gene from L. (L.) amazonensis. Multiple protein sequence alignment of all known trypanosomatid TRs highlights the high degree of conservation and illustrates the phylogenetic relationships. A 3D homology model for L. amazonensis TR was constructed based on the previously reported Crithidia fasciculata structure. The purified recombinant TR shows enzyme activity and in vivo expression of the native enzyme could be detected in infective promastigotes, both by Western blotting and by immunofluorescence.

[Analysis of polymorphisms in the trypanothione reductase and cruzipain genes in Colombian strains of Trypanosoma cruzi]. / Análisis de polimorfismos en los genes tripanotión reductasa y cruzipaína en cepas colombianas de Trypanosoma cruzi.

INTRODUCTION: Genetic studies of Trypanosoma cruzi have tried to establish relations between genetic variants and their biological characteristics, such as clinical manifestations, host or geographic origin. However, much controversy exists on the associations between the commonly used DNA markers with group, clinical characteristics and disease epidemiology. OBJECTIVE: In this study determined the variability of the genes that code for the proteins trypanothione reductase and cruzipain, both involved in the infection and survival of the parasite in the mammalian host, was studied and the association between genetic polymorphism and biological and geographic sources in Colombian T. cruzi strains was examined. MATERIALS AND METHODS: The genotypes for each of six SNPs (single nucleotide polymorphisms) for trypanothione reductase and eight SNPs for cruzipain genes were identified by polymerase chain reaction-restriction fragment length polymorphism in 36 T. cruzi Colombian stocks from several regions and biological origins. RESULTS: Three genotypes were identified for trypanothione reductase with Acy I and Hae III enzymes and six genotypes for cruzipain with the Rsa I, Ban I and Bsu 361 enzymes. CONCLUSIONS: For trypanothione reductase, an association was not established with biological or geographical origin; however, alleles at positions 102 and 210 allowed discrimination with groups I and II. For cruzipain, specific genotypes were associated with group, biological and geographic origin. The usefulness of molecular markers on these genes was demonstrated for the determination and differentiation of genetic varieties in T. cruzi.

Protein disulfide oxidoreductases and the evolution of thermophily: was the last common ancestor a heat-loving microbe?

Protein disulfide oxidoreductases (PDOs) are redox enzymes that catalyze dithiol-disulfide exchange reactions. Their sequences and structure reveal the presence of two thioredoxin fold units, each of which is endowed with a catalytic site CXXC motif. PDOs are the outcome of an ancient gene duplication event. They have been described in a number of thermophilic and hyperthermophilic species, where they play a critical role in the structural stabilization of intracellular proteins. PDOs are homologous to both the N-terminal domain of the bacterial alkyl hydroperoxide reductase (AhpF) and to the eukaryotic protein disulfide isomerase (PDI). Phylogenetic analysis of PDOs suggests that they first evolved in the crenarchaeota, spreading from them into the Bacteria via the euryarchaeota. These results imply that the last common ancestor (LCA) of all extant living beings lacked a PDO and argue, albeit weakly, against a thermophilic LCA.