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Biomedical subjects

Pedro L Oliveira

Publications and source records attributed to Pedro L Oliveira.

At least 19 recordsLinked to original sources
PubMed2006

Oxidative stress impairs heme detoxification in the midgut of the cattle tick, Rhipicephalus (Boophilus) microplus.

In the cattle tick Rhipicephalus (Boophilus) microplus digestion of blood is intracellular, accomplished by the so-called digest cells that fill the midgut lumen. Hydrolysis of hemoglobin in the digestive vesicles of these cells results in the release of large amounts of heme, a pro-oxidant compound, whose iron atom, together with H(2)O(2), may participate in the Fenton reaction and lead to the production of hydroxyl radicals. Here, we investigated the role of catalase, an enzyme responsible for H(2)O(2) detoxification. Fully engorged female ticks injected with 3-amino-1,2,4-triazole (AT), a catalase inhibitor, showed increased H(2)O(2) in the gut, together with diminished life span and lower egg-laying rates. Increased mortality observed upon AT injection was reversed by further injection of exogenous catalase, 2 days after AT treatment, confirming that increased death was due to inhibition of this enzyme by AT. In primary cultures of digest cells, intracellular H(2)O(2) is limited to specific organelles, while treatment with AT in vitro resulted in increased H(2)O(2) spreading all over the cell, confirming the role of catalase in regulating H(2)O(2) levels. Ticks fed on a calf that had been injected with AT showed marked inhibition of catalase activity in the gut and diminished life span, oviposition and engorgement. Digest cells of these ticks had an altered morphology, showing heme spread all over the cytosol, instead of being limited to the hemosomes. The amount of aggregated heme found in isolated hemosome was also strongly decreased in AT-treated cattle. All together, our results indicate that catalase performs an important role in the control of redox balance in R. microplus, which dramatically affects hemosome formation and stability. This enzyme may be a target in the development of new methods for tick control.

Amitrole↗
PubMed2006

Vaccination of bovines with recombinant Boophilus Yolk pro-Cathepsin.

Boophilus Yolk pro-Cathepsin (BYC) is an aspartic proteinase found in Boophilus microplus eggs that is involved in the embryogenesis and has been tested as antigen to compose an anti-tick vaccine. The vaccine potential of a recombinant BYC expressed in Escherichia coli (rBYC) was investigated. rBYC was purified and used to immunize Hereford cattle. The sera of bovines immunized with rBYC recognized the native BYC with a titer ranging from 125 to 4000. Furthermore, immunized bovines challenged with 20,000 larvae presented an overall protection of 25.24%. The partial protection obtained against B. microplus infestation with the recombinant protein immunization was similar to the already described for native BYC immunization.

Animals↗
PubMed2006

Identification of the Aedes aegypti peritrophic matrix protein AeIMUCI as a heme-binding protein.

The gene Aedes aegypti intestinal mucin 1 (AeIMUC1) encodes a putative peritrophic matrix (PM) protein that is expressed in the midgut of mosquito larvae and adults and is upregulated in response to exposure to heavy metals. The AeIMUC1 protein has a predicted secretory signal peptide and three putative chitin-binding domains (CBDs) with an intervening mucin-like domain. Immunofluorescence and immunoelectron microscopy experiments established that AeIMUC1 is a bona fide PM protein, and binding of the recombinant protein to chitin was demonstrated in vitro. Previous experiments suggested that the Ae. aegypti PM can bind toxic heme molecules generated during blood digestion. However, the identity of the binding molecule(s) was unknown. Using of heme-agarose beads and spectrophotometric and microcalorimetric titrations, we show that recombinant AeIMUC1 can bind large amounts of heme in vitro, suggesting for the first time a role for a PM protein in heme detoxification during blood digestion. Binding of heme to AeIMUC1 was accompanied by an altered circular dichroism spectrum indicating a change in protein conformation, consistent with an increase in secondary structure. Heme-binding activity was mapped to the AeIMUC1 CBDs, suggesting that these domains possess dual chitin- and heme-binding activity.

Aedes↗
PubMed2006

A heme-degradation pathway in a blood-sucking insect.

Hematophagous insects are vectors of diseases that affect hundreds of millions of people worldwide. A common physiological event in the life of these insects is the hydrolysis of host hemoglobin in the digestive tract, leading to a massive release of heme, a known prooxidant molecule. Diverse organisms, from bacteria to plants, express the enzyme heme oxygenase, which catalyzes the oxidative degradation of heme to biliverdin (BV) IX, CO, and iron. Here, we show that the kissing bug Rhodnius prolixus, a vector of Chagas' disease, has a unique heme-degradation pathway wherein heme is first modified by addition of two cysteinylglycine residues before cleavage of the porphyrin ring, followed by trimming of the dipeptides. Furthermore, in contrast to most known heme oxygenases, which generate BV IXalpha, in this insect, the end product of heme detoxification is a dicysteinyl-BV IXgamma. Based on these results, we propose a heme metabolizing pathway that includes the identified intermediates produced during modification and cleavage of the heme porphyrin ring.

Animals↗
PubMed2006

Adaptations against heme toxicity in blood-feeding arthropods.

A blood-sucking habit appeared independently several times in the course of arthropod evolution. However, from more than a million species of insects and arachnids presently living on earth, only about 14,000 species developed the capacity to feed on vertebrate blood. This figure suggests the existence of severe physiological constraints for the evolution of hematophagy, implying the selective advantage of special adaptations related to the use of blood as a food source. Digestion of vertebrate hemoglobin in the midgut of blood-feeding arthropods results in the production of large amounts of heme, a potentially cytotoxic molecule. Here we will review mechanisms by which heme can exert biological damage, together with a wide spectrum of adaptations developed by blood-feeding insects and ticks to counteract its deleterious effects. In spite of the existence of a great molecular diversity of protective mechanisms, different hematophagous organisms developed convergent solutions that may be physiologically equivalent.

Adaptation, Physiological↗
PubMed2005

Structural and morphological characterization of hemozoin produced by Schistosoma mansoni and Rhodnius prolixus.

Hemozoin (Hz) is a heme crystal produced upon the digestion of hemoglobin (Hb) by blood-feeding organisms as a main mechanism of heme disposal. The structure of Hz consists of heme dimers bound by reciprocal iron-carboxylate interactions and stabilized by hydrogen bonds. We have recently described heme crystals in the blood fluke, Schistosoma mansoni, and in the kissing bug, Rhodnius prolixus. Here, we characterized the structures and morphologies of the heme crystals from those two organisms and compared them to synthetic beta-hematin (betaH). Synchrotron radiation X-ray powder diffraction showed that all heme crystals share the same unit cell and structure. The heme crystals isolated from S. mansoni and R. prolixus consisted of very regular units assembled in multicrystalline spherical structures exhibiting remarkably distinct surface morphologies compared to betaH. In both organisms, Hz formation occurs inside lipid droplet-like particles or in close association to phospholipid membranes. These results show, for the first time, the structural and morphological characterization of natural Hz samples obtained from these two blood-feeding organisms. Moreover, Hz formation occurring in close association to a hydrophobic environment seems to be a common trend for these organisms and may be crucial to produce very regular shaped phases, allowing the formation of multicrystalline assemblies in the guts of S. mansoni and R. prolixus.

Animals↗
PubMed2005

Purification and antigenicity of two recombinant forms of Boophilus microplus yolk pro-cathepsin expressed in inclusion bodies.

The tick Boophilus microplus is a bovine ectoparasite present in tropical and subtropical areas of the world and the use of vaccines is a promising method for tick control. BYC is an aspartic proteinase found in eggs that is involved in the embryogenesis of B. microplus and was proposed as an important antigen in the development of an anti-tick vaccine. The cDNA of BYC was amplified by PCR and cloned for expression in two forms with and without thioredoxin fusion protein (Trx), coding recombinant proteins named rBYC-Trx and rBYC, respectively. Expression, solubility, and yields of the two forms were analyzed. The recombinant proteins were expressed in inclusion bodies (IBs) and three denaturant agents (N-lauroyl sarcosine, guanidine hydrochloride, and urea) were tested for IBs solubilization. The N-lauroyl sarcosine solubilized 90.4 and 92.4% of rBYC-Trx and rBYC IBs, respectively, and was the most efficient denaturant. Two recombinant forms were affinity-purified by Ni2+-Sepharose under denaturing conditions. After dialysis, the yield of soluble protein was 84.1% for r-BYC-Trx and 5.9% for rBYC. These proteins were immune-reactive against sera from rabbit, mouse, and bovine previously immunized with native BYC, which confirms the antigenicity of the recombinant BYCs expressed in the Escherichia coli system.

Animals↗
PubMed2004

Inhibition of heme aggregation by chloroquine reduces Schistosoma mansoni infection.

Adult Schistosoma mansoni digest large amounts of host hemoglobin and release potentially toxic heme inside their guts. We have previously demonstrated that free heme in S. mansoni is detoxified through aggregation, forming hemozoin (Hz). Possible mechanisms of heme aggregation and the effects of chloroquine (CLQ) on formation of Hz and on the viability of this parasite have now been investigated. Different fractions isolated from S. mansoni, such as crude whole-worm homogenates, total lipid extracts, and Hz itself promoted heme aggregation in vitro in a CLQ-sensitive manner. Treatment of S. mansoni-infected mice with CLQ led to remarkable decreases in total protein, Hz content, and viability of the worms, as well as in parasitemia and deposition of eggs in mouse livers. These results indicate that inhibition of formation of Hz in S. mansoni, by CLQ, led to an important decrease in the overall severity of experimental murine schistosomiasis. Taken together, the results presented here suggest that formation of Hz is a major mechanism of heme detoxification and a potential target for chemotherapy in S. mansoni.

Animals↗
PubMed2004

HeLp, a heme-transporting lipoprotein with an antioxidant role.

Plasma lipoproteins involved in lipid transport are target for free radical-evoked pathological conditions in several mammalian models. The main hemolymphatic protein of Boophilus microplus is a heme-binding lipoprotein (HeLp, for Heme LipoProtein) that carries dietary heme produced from degradation of vertebrate hemoglobin to tissues of the tick. Addition of heme to phospholipid liposomes resulted in intense lipid peroxidation, which was inhibited by addition of HeLp. HeLp prevented lysis of red blood cells by heme. HeLp also inhibited reactions of heme with tert-butyl hydroperoxide (t-BOOH) or hydrogen peroxide. HeLp, quite differently from other lipoproteins, presents a protective intrinsic mechanism to counteract heme toxicity, while preserving the heme molecule to be reused by the tick. This is the first report of a lipoprotein acting as an antioxidant particle against heme-induced radical damage.

Animals↗
PubMed2004

Proteolytic activity of Boophilus microplus Yolk pro-Cathepsin D (BYC) is coincident with cortical acidification during embryogenesis.

In a previous report (Parasitology 116 (1998) 525) we isolated and characterized Boophilus Yolk pro-Cathepsin (BYC), an aspartic proteinase precursor from the eggs of the hard tick. The present study was designed to characterize the function of BYC in the consumption of vitellin (VT), the major yolk protein, during embryogenesis. Both purified BYC and total egg homogenate proteolytic activity showed a similar pH dependence profile with an acidic optimum. Purified BYC presented higher activity against VT as a substrate when compared to other proteins. The VT degradation pattern observed in vitro also showed a similar profile to that observed in vivo. Co-localization of BYC and acidic cortical yolk granules was performed by immunocytochemistry and confocal microscopy. Proton-pumping activity of yolk granules in vitro was higher in eggs collected 4 day after oviposition than in newly laid eggs. Taken together, our data suggest that BYC plays a major role in the degradation of VT and that its activity is controlled by acidification of yolk platelets localized at the cortical cytoplasm of the developing Boophilus microplus egg.

Animals↗
PubMed2003

Changes in salivary nitrophorin profile during the life cycle of the blood-sucking bug Rhodnius prolixus.

The insect Rhodnius prolixus is a hematophagous hemipteran that has five nymphal instars. Fifth instar nymphs contain, in their salivary glands, four nitrophorins which have already been described in the literature (NP1, NP2, NP3 and NP4). Two new hemeproteins were isolated and partially characterized from first instar nymphs. NP2, that shows an anticoagulant activity, was also identified, but NP1, NP3 and NP4 were not found. As these new hemeproteins have amino-terminal sequences clearly homologous to already described nitrophorins and were capable of binding nitric oxide, they were named nitrophorins 5 and 6, although they showed an unusual Soret band at 412 nm. In each subsequent nymphal stage, a new nitrophorin emerges. In the second instar, NP4 comes into view, in the third instar NP1 appears, and NP3 is only found in fifth instar nymphs and adults, showing that the nitrophorin profile of R. prolixus saliva is stage-specific.

Amino Acid Sequence↗
PubMed2003

A new intracellular pathway of haem detoxification in the midgut of the cattle tick Boophilus microplus: aggregation inside a specialized organelle, the hemosome.

The hard tick Boophilus microplus ingests large volumes of cattle blood, as much as 100 times its own mass before feeding. Huge amounts of haem are produced during haemoglobin digestion, which takes place inside acidic lysosomal-type vacuoles of the digest cells of the midgut. Haem is a promoter of free radical formation, so haemoglobin digestion poses an intense oxidative challenge to this animal. In the present study we followed the fate of the haem derived from haemoglobin hydrolysis in the digest cells of the midgut of fully engorged tick females. The tick does not synthesize haem, so during the initial phase of blood digestion, absorption is the major route taken by the haem, which is transferred from the digest cells to the tick haemocoel. After this absorptive period of a few days, most of the haem produced upon haemoglobin degradation is accumulated in the interior of a specialized, membrane-delimited, organelle of the digest cell, herein called hemosome. Haem accounts for 90% of the hemosome mass and is concentrated in the core of this structure, appearing as a compact, non-crystalline aggregate of iron protoporphyrin IX without covalent modifications. The unusual FTIR spectrum of this aggregate suggests that lateral propionate chains are involved in the association of haem molecules with other components of the hemosome, which it is proposed is a major haem detoxification mechanism in this blood-sucking arthropod.

Animals↗
PubMed2002

Vampires, Pasteur and reactive oxygen species. Is the switch from aerobic to anaerobic metabolism a preventive antioxidant defence in blood-feeding parasites?

Several species of parasites show a reduction of their respiratory activity along their developmental cycles after they start to feed on vertebrate blood, relying on anaerobic degradation of carbohydrates to achieve their energy requirements. Usually, these parasites choose not to breathe despite of living in an environment of high oxygen availability such as vertebrate blood. Absence of the 'Pasteur effect' in most of these parasites has been well documented. Interestingly, together with the switch from aerobic to anaerobic metabolism in these parasites, there is clear evidence pointing to an increase in their antioxidant defences. As the respiratory chain in mitochondria is a major site of production of reactive oxygen species (ROS), we propose here that the arrest of respiration constitutes an adaptation to avoid the toxic effects of ROS. This situation would be especially critical for blood-feeding parasites because ROS produced in mitochondria would interact with pro-oxidant products of blood digestion, such as haem and/or iron, and increase the oxidative damage to the parasite's cells.

Anaerobiosis↗
PubMed2002

Neutrophil activation by heme: implications for inflammatory processes.

Heme, a ubiquitous iron-containing compound, is present in large amounts in many cells and is inherently dangerous, particularly when it escapes from intracellular sites. The release of heme from damaged cells and tissues is supposed to be higher in diseases such as malaria and hemolytic anemia or in trauma and hemorrhage. We investigated here the role of free ferriprotoporphyrin IX (hemin) as a proinflammatory molecule, with particular attention to its ability to activate neutrophil responses. Injecting hemin into the rat pleural cavity resulted in a dose-dependent migration of neutrophils, indicating that hemin is able to promote the recruitment of these cells in vivo. In vitro, hemin induced human neutrophil chemotaxis and cytoskeleton reorganization, as revealed by the increase of neutrophil actin polymerization. Exposure of human neutrophils to 3 microM hemin activated the expression of the chemokine interleukin-8, as demonstrated by quantitative reverse-transcription polymerase chain reaction, indicating a putative molecular mechanism by which hemin induces chemotaxis in vivo. Brief incubation of human neutrophils with micromolar concentrations of hemin (1-20 microM) triggered the oxidative burst, and the production of reactive oxygen species was directly proportional to the concentration of hemin added to the cells. Finally, we observed that human neutrophil protein kinase C was activated by hemin in vitro, with a K(1/2) of 5 microM. Taken together, these results suggest a role for hemin as a proinflammatory agent able to induce polymorphonuclear neutrophil activation in situations of clinical relevance, such as hemolysis or hemoglobinemia.

Actins↗
PubMed2002

On the pro-oxidant effects of haemozoin.

Haemozoin (Hz) is a haem aggregate produced in some blood-feeding organisms. There is a general belief that Hz formation would be a protective mechanism against haem toxicity. Here we show that when aggregated into Hz, haem is less deleterious than its free form. When haem was added to phosphatidylcholine (PC) liposomes, there was an intense stimulation of oxygen consumption, which did not occur when Hz was incubated with the same preparation. Evaluation of oxygen radical attack to lipids, by measurement of thiobarbituric acid reactive substances (TBARS), showed significantly lower levels of lipid peroxidation in samples containing PC liposomes incubated with Hz than with haem. However, TBARS production induced by Hz was much higher when using 2-deoxyribose (2-DR) as substrate, than with PC liposomes. Spin-trapping analysis by electron paramagnetic resonance (EPR) of Hz and tert-butylhydroperoxide (tert-BuOOH) showed that production of methoxyl and tert-butoxyl radicals was only slightly reduced compared to what was observed with haem. Interestingly, when large Hz crystals were used in 2-DR TBARS assays and tert-BuOOH EPR experiments, the pro-oxidant effects of Hz were strongly reduced. Moreover, increasing concentrations of Hz did not induce erythrocyte lysis, as occurred with haem. Thus, the reduced capacity of Hz to impose radical damage seems to result from steric hindrance of substrates to access the aggregated haem, that becomes less available to participate in redox reactions.

Animals↗
PubMed2002

Aedes aegypti peritrophic matrix and its interaction with heme during blood digestion.

A large amount of heme is produced upon digestion of red cell hemoglobin in the midgut of mosquitoes. The interaction between heme and the peritrophic matrix (PM) was studied in Aedes aegypti. By light microscopy, the PM appeared as a light brownish layer between the intestinal epithelium and the alimentary bolus. This natural color can be attributed to the presence of heme bound to the matrix. In histochemical studies, a diffuse peroxidase activity of the heme molecules was clearly observed between the erythrocytes and the PM at 14 h after the blood meal. This activity tends to increase and concentrate in the PM reaching its maximum thickness at 24 h after feeding. Most of the heme of the PM was found associated to with enormous number of small electron-dense granules. The amount of heme bound to the PM increased in parallel with the progression of digestion, reaching a maximum at 48 h after feeding, when 18 nmol of heme were found in an individual matrix. The association of heme with PM from insects fed with plasma is saturable, suggesting the existence of specific binding sites for hemin in the PM. Taken all together, our data indicate that the PM performs a central role in heme detoxification in this insect.

Aedes↗
PubMed2002

Rhodnius heme-binding protein (RHBP) is a heme source for embryonic development in the blood-sucking bug Rhodnius prolixus (Hemiptera, Reduviidae).

We have previously shown that Rhodnius prolixus' eggs and hemolymph are pink due to the presence of the hemeprotein Rhodnius heme-binding protein (RHBP). In the hemolymph it functions as an antioxidant. Nevertheless, its function in eggs has not been determined. Here we present evidence that RHBP is a source of heme for embryonic development. RHBP content decreases during embryogenesis, but the total heme content of eggs remains unchanged. Biliverdin, the product of heme degradation, is not detectable in late embryos. The activity of the heme-synthesizing pathway is low throughout embryogenesis and rises sharply after nymphs' hatching. Heme-radiolabeled eggs were produced and, at the day of hatching, nymphs were dissected. The presence of radiolabeled heme in their carcass is an indication that heme reutilization is occurring. The only animal known to reutilize heme in significant levels is the cattle tick Boophilus microplus, which cannot synthesize its own heme. Diversely, Rhodnius can synthesize its own heme but, in the context of embryogenesis, heme demand seems to be supplied by the programmed release of heme form RHBP. This behavior indicates that in Rhodnius, we might have a highly unusual profile: heme is both synthesized and reutilized.

Animals↗