The American trypanosome Trypanosoma cruzi is exposed to toxic oxygen metabolites that are generated by drug metabolism and immune responses in addition to those produced by endogenous processes. ...However, much remains to be resolved about the parasite oxidative defense system, including the mechanism(s) of peroxide reduction. Here we show that reduction of peroxides inT. cruzi is catalyzed by two distinct trypanothione-dependent enzymes. These were localized to the cytosol and mitochondrion. Both are members of the peroxiredoxin family of antioxidant proteins and are characterized by the presence of two conserved domains containing redox active cysteines. The role of these proteins in protecting T. cruzi from peroxide-mediated damage was demonstrated following overexpression of enzyme activity. The parasite-specific features of T. cruzicytoplasmic peroxiredoxin and T. cruzi mitochondrial peroxiredoxin may be exploitable in terms of drug development.
Background: There is limited evidence on whether particulate matter (PM) can augment the progression of atherosclerosis; furthermore, the specific attributes of PM responsible for health effects are ...unclear. We developed models to predict exposure to PM <10 μm (PM 10 ) and also to predict a measure of oxidative potential (the capacity of particles to induce oxidative damage). Our objectives were (1) to estimate the association between PM 10 and carotid intima-media thickness, a measure of subclinical atherosclerosis, and (2) to compare this association with that of PM 10 weighted by its oxidative potential (PM 10 *OP) Methods: Analysis was based on 2348 participants of the Whitehall II cohort of British civil servants who had intima-media thickness measured between 2003 and 2005 and lived in Greater London. Weekly PM 10 and PM 10 *OP were predicted at each participant's residence. Primary exposure metrics were defined as PM10 and PM 10 *OP averaged over the year before scan. We estimated associations between exposure metrics and intima-media thickness using generalized linear regression models. Results: An interquartile range increase (5.2 μm —3 ) in PM10 was associated with a 5.0% (95% confidence interval = 1.9% to 8.3%) increase in intima-media thickness after covariate adjustment. The association for an interquartile range change in PM 10 *OP (1.5 m —3 ) was weaker: 1.2% (0.2% to 2.2%). Conclusions: These findings support a relationship between PM exposure and atherosclerosis. PM weighted by this particular measure of oxidative potential was not more predictive of the extent of atherosclerosis than PM mass concentration.
Rapid, semiautomated, and fully automated multiplex real-time RT-PCR assays were developed and validated for the detection of influenza (Flu) A, Flu B, and respiratory syncytial virus (RSV) from ...nasopharyngeal specimens. The assays can detect human H1N1, H3N2, and swine-origin (S-OIV) H1N1 Flu A viruses and were effectively used to distinguish Flu A infections (of all subtypes) from Flu B and RSV infections during the current S-OIV outbreak in Milwaukee, WI. The analytical limits of detection were 10−2 to 101 TCID50 /ml depending on the platform and analyte and showed only one minor cross-reaction among 23 common respiratory pathogens (intermittent cross-reaction to adenovirus at >107 TCID50 /ml). A total of 100 clinical samples were tested by tissue culture, both automated assays, and the US Food and Drug Administration-approved ProFlu+ assay. Both the semiautomated and fully automated assays exhibited greater overall (Flu A, Flu B, and RSV combined) clinical sensitivities (93 and 96%, respectively) and individual Flu A sensitivities (100%) than the Food and Drug Administration-approved test (89% overall sensitivity and 93% Flu A sensitivity). All assays were 99% specific. During the S-OIV outbreak in Milwaukee, WI, the fully automated assay was used to test 1232 samples in 2 weeks. Flu A was detected in 134 clinical samples (126 H1N1 S-OIV, 5 H1N1 human, and 1 untyped) with 100% positive agreement compared with other “in-house” validated molecular assays, with only 2 false-positive results. Such accurate testing using automated high-throughput molecule systems should allow clinicians and public health officials to react quickly and effectively during viral outbreaks.
Neovascularization in the retina and iris of diabetic patients is a major cause of severe visual loss. However, study of these lesions is compromised by the lack of a comparable diabetic rodent ...model. Because the vasoactive and angiogenic agent, angiotensin II, is involved in diabetic microvascular disease, we aimed to determine whether endothelial cell proliferation could be induced in the retinae and irides of hypertensive transgenic (mRen-2)27 rats that display an enhanced extra-renal renin-angiotensin system (RAS), including the eye. Six-week-old Ren-2, spontaneously hypertensive, and Sprague-Dawley rats received either streptozotocin or control vehicle and were studied for 36 weeks. Additional nondiabetic and diabetic Ren-2 rats were treated throughout with the angiotensin-converting enzyme inhibitor lisinopril (LIS) (10 mg/kg/day in drinking water). Endothelial cell proliferation was only observed in retinae and irides of diabetic Ren-2 rats and was reduced with LIS. In diabetic Ren-2, vascular endothelial growth factor (VEGF) and VEGFR-2 mRNA were increased in retinae and irides and reduced with LIS. Diabetes activated ocular renin in Ren-2 but not Sprague-Dawley rats. The diabetic Ren-2 rat is a model of intraocular endothelial cell proliferation that can be attenuated by RAS blockade via VEGF-dependent pathways. RAS blockade is a potential treatment for vision-threatening diabetic microvascular complications.
Background. Osteopontin is a macrophage chemotactic protein that has been pathogenetically linked to tissue injury in non‐diabetic kidney disease. Methods. To examine osteopontin expression and ...macrophage accumulation in diabetic nephropathy, diabetes was induced with streptozotocin (STZ) in the transgenic (mRen‐2)27 rat, a rodent model which develops the structural and functional features of its human counterpart when rendered diabetic. Non‐diabetic rats were randomly selected to receive either (STZ) or citrate buffer. Diabetic rats were further randomly selected to receive either the angiotensin‐converting‐enzyme inhibitor, perindopril (6 mg/kg/day), or the vehicle only for 12 weeks. Results. When compared with control animals, diabetes was associated with a 10‐fold increase in the gene expression of osteopontin. Increased transcript and immunostainable osteopontin were detected in tubular epithelial cells in association with extensive macrophage accumulation. Treatment with perindopril significantly ameliorated the overexpression of osteopontin in association with attenuation of macrophage accumulation. Conclusions. These findings suggest that osteopontin expression and macrophage accumulation may play a role in the tubulointerstitial injury in diabetic nephropathy, and that inhibition of osteopontin expression may be one of the mechanisms by which blockade of the renin‐angiotensin system confers a renoprotective effect.
ObjectivesFirst, we present a general analytical approach to estimating the association between medium-term changes in air pollution and health across small areas. As a specific illustration, we then ...applied the approach to data on London residents from a 4-year period to test whether reductions in traffic-related air pollution were associated with reductions in cardio-respiratory hospital admissions.MethodsA binomial distribution was used to model change in admissions over time in each small area, which was measured as the proportion of admissions in 2003–2004 out of admissions over all study years (2001–2004). Annual average concentrations of nitrogen oxides (NOx) were modelled using an emissions-dispersion model. The association between change in NOx and change in hospital admissions was estimated using logistic regression and an instrumental variable approach.ResultsFor some diagnostic groups, suggestive associations between reductions in NOx and reductions in admissions were observed, for example, OR=0.97 (95% CI 0.96 to 0.99) for an IQR decrease in NOx (3 μg/m3) and all respiratory admissions. Accounting for spatial dependence attenuated several of the associations; for respiratory admissions, the OR was 1.00 (95% CI 0.98 to 1.02), leaving only that for bronchiolitis significant (OR=0.91; 95% CI 0.84 to 0.99). In this particular illustration, the instrumental variable approach did not appear to add information.ConclusionsIn this illustration, there was relatively limited power to detect an association between changes in air pollution and hospital admissions over time. However, the analytical approach could deliver more robust estimates of the health effects of changes in air pollution in settings with greater spatial contrast in changes in air pollution over time.
Reactive oxygen species are the unwanted by-products of aerobic metabolism. To protect cells against their potentially lethal effects a series of pathways have evolved that are collectively called ...the oxidative defence system. In most eukaryotes, catalases and selenium-dependent glutathione peroxidases form the front line of defence against hydroperoxide-mediated damage. However, these activities are lacking in members of the Trypanosomatidae family of protozoan parasites. Instead these organisms contain several enzyme-mediated pathways for removal of hydroperoxides that are centred upon the unusual thiol trypanothione. Here we discuss the biochemical properties of one group of these enzymes, the non-selenium glutathione-dependent peroxidases, and outline the roles that they play in protecting the parasite against hydroperoxides associated with biological membranes.
In most eukaryotes, glutathione-dependent peroxidases play a key role in the metabolism of peroxides. Numerous studies have reported that trypanosomatids lack this activity. Here we show that this is ...not the case, at least for the American trypanosome Trypanosoma cruzi. We have isolated a single-copy gene from T. cruzi with the potential to encode an 18 kDa enzyme, the sequence of which has highest similarity with glutathione peroxidases from plants. A recombinant form of the protein was purified following expression in Escherichia coli. The enzyme was shown to have peroxidase activity in the presence of glutathione/glutathione reductase but not in the presence of trypanothione/trypanothione reductase. It could metabolize a wide range of hydroperoxides (linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide>cumene hydroperoxide>t-butyl hydroperoxide), but no activity towards hydrogen peroxide was detected. Enzyme activity could be saturated by glutathione when both fatty acid and short-chain organic hydroperoxides were used as substrate. For linoleic acid hydroperoxide, the rate-limiting step of this reaction is the reduction of the peroxidase by glutathione. With lower-affinity substrates such as t-butyl hydroperoxide, the rate-limiting step is the reduction of the oxidant. The data presented here identify a new arm of the T. cruzi oxidative defence system.
Airway surface hydration depends on the balance between transepithelial Na(+) absorption and Cl(-) secretion. In adult mice, absence of functional cystic fibrosis transmembrane conductance regulator ...(Cftr) fails to recapitulate human cystic fibrosis (CF) lung disease. In contrast, overexpression of the epithelial Na(+) channel β subunit in transgenic mice (βENaC-Tg) produces unregulated Na(+) hyperabsorption and results in CF-like airway surface dehydration, mucus obstruction, inflammation, and increased neonatal mortality. To investigate whether the combination of airway Na(+) hyperabsorption and absent Cftr-mediated Cl(-) secretion resulted in more severe lung pathology, we generated double-mutant ΔF508 CF/βENaC-Tg mice. Survival of ΔF508 CF/βENaC-Tg mice was reduced compared with βENaC-Tg or ΔF508 CF mice. Absence of functional Cftr did not affect endogenous or transgenic ENaC currents but produced reduced basal components of Cl(-) secretion and tracheal cartilaginous defects in both ΔF508 CF and ΔF508 CF/βENaC-Tg mice. Neonatal ΔF508 CF/βENaC-Tg mice exhibited higher neutrophilic pulmonary inflammation and club cell (Clara cell) necrosis compared with βENaC-Tg littermates. Neonatal ΔF508 CF/βENaC-Tg mice also exhibited spontaneous bacterial infections, but the bacterial burden was similar to that of βENaC-Tg littermates. Adult ΔF508 CF/βENaC-Tg mice exhibited pathological changes associated with eosinophilic crystalline pneumonia, a phenotype not observed in age-matched βENaC-Tg mice. Collectively, these data suggest that the combined abnormalities in Na(+) absorption and Cl(-) secretion produce more severe lung disease than either defect alone. Airway cartilage abnormalities, airway cell necrosis, and exaggerated neutrophil infiltration likely interact with defective mucus clearance caused by βENaC overexpression and absent CFTR-mediated Cl(-) secretion to produce the increased neonatal mortality observed in ΔF508 CF/βENaC-Tg mice.
The PEB1a protein is an antigenic factor exposed on the surface of the food-borne human pathogen
Campylobacter jejuni, which has a major role in adherence and host colonisation. PEB1a is also the ...periplasmic binding protein component of an aspartate/glutamate ABC transporter essential for optimal microaerobic growth on these dicarboxylic amino acids. Here, we report the crystal structure of PEB1a at 1.5 Å resolution. The protein has a typical two-domain α/β structure, characteristic of periplasmic extracytoplasmic solute receptors and a chain topology related to the type II subfamily. An aspartate ligand, clearly defined by electron density in the interdomain cleft, forms extensive polar interactions with the protein, the majority of which are made with the larger domain. Arg89 and Asp174 form ion-pairing interactions with the main chain α-carboxyl and α-amino-groups, respectively, of the ligand, while Arg67, Thr82, Lys19 and Tyr156 co-ordinate the ligand side-chain carboxyl group. Lys19 and Arg67 line a positively charged groove, which favours binding of Asp over the neutral Asn. The ligand-binding cleft is of sufficient depth to accommodate a glutamate. This is the first structure of an ABC-type aspartate-binding protein, and explains the high affinity of the protein for aspartate and glutamate, and its much weaker binding of asparagine and glutamine. Stopped-flow fluorescence spectroscopy indicates a simple bimolecular mechanism of ligand binding, with high association rate constants. Sequence alignments and phylogenetic analyses revealed PEB1a homologues in some Gram-positive bacteria. The alignments suggest a more distant homology with GltI from
Escherichia coli, a known glutamate and aspartate-binding protein, but Lys19 and Tyr156 are not conserved in GltI. Our results provide a structural basis for understanding both the solute transport and adhesin/virulence functions of PEB1a.
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