Urate synthesis in the blood-sucking insect rhodnius prolixus. Stimulation by hemin is mediated by protein kinase C.
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Hemin is a catalyst of the formation of reactive oxygen species. We proposed that hematophagous insects are exposed to intense oxidative stress because of hemoglobin hydrolysis in their midgut (Petretsky, M. D., Ribeiro, J. M. C., Atella, G. C., Masuda, H., and Oliveira, P. L. (1995) J. Biol. Chem. 270, 10893-10896). We have shown that hemin stimulates urate synthesis in the blood-sucking insect Rhodnius prolixus (Graca-Souza, A. V., Petretsky, J. H., Demasi, M., Bechara, E. J. H., and Oliveira, P. L. (1997) Free Radical Biol. Med. 22, 209-214). Once released by fat body cells, urate accumulates in the hemolymph, where this radical scavenger constitutes an important defense against blood-feeding derived oxidative stress. Incubation of Rhodnius fat bodies with okadaic acid raises the level of urate synthesis, suggesting that urate production can be controlled by protein phosphorylation/dephosphorylation. Urate synthesis is stimulated by dibutyryl cAMP and inhibited by N(2((p-bromocinnamil)amino)ethyl)-5-isoquinolinesulfonamide (H-89), an inhibitor of protein kinase A, as well as activated by the protein kinase C activator phorbol 12-myristate 13-acetate. In the presence of hemin, however, inhibition of urate synthesis by H-89 does not occur, suggesting that the hemin stimulatory effect is not mediated by protein kinase A. Calphostin C completely inhibits the hemin-induced urate production, suggesting that the triggering of urate antioxidant response depends on protein kinase C activation. This conclusion is reinforced by the observation that in fat bodies exposed to hemin, both protein kinase C activity and phosphorylation of specific endogenous polypeptides are significantly increased. (+info)
Bednet impregnation for Chagas disease control: a new perspective.
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BACKGROUND: To determine the efficacy and acceptability of deltamethrin-impregnated bednets in controlling Chagas disease in South America. METHODS: In three endemic departments of Colombia, a qualitative study on people's knowledge about Chagas disease, vectors, preventive measures and their willingness for collaboration in control operations was undertaken. Additionally, in an entomological study with 100 laboratory-bred Chagas vectors (Rhodnius prolixus), vectors were released for 5 nights (20 each night) in an experimental room, with the human bait protected for one night by an unimpregnated and for four nights by a deltamethrin-impregnated bednet (13 mg/m2). Vectors were stained with fluorescent powder for observation, collected after 10 h exposure in the experimental room and observed for a further 72 h. RESULTS: The study population did not know anything about Chagas disease, but believed the vector to transmit cutaneous leishmaniasis. Therefore willingness to take part in control operations was high. The experimental hut study showed a vector mortality rate of 95% in a room with impregnated nets and of 10% in a room with unimpregnated nets. CONCLUSION: This study opens a new perspective for Chagas disease control in integrated vector borne disease prevention programmes. (+info)
Utility of the polymerase chain reaction in detection of Trypanosoma cruzi in Guatemalan Chagas' disease vectors.
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For effective control programs, accurate assessment of Trypanosoma cruzi infection in vectors is essential and has traditionally been performed by microscopic examination. For particular vectors and not others, polymerase chain reaction (PCR) analysis of fecal samples recently has been shown to be an effective means of detection. The sensitivities of the PCR and microscopy for detection of T. cruzi in different anatomic sites were compared in the two major vectors of Guatemala, Triatoma dimidiata and Rhodnius prolixus. Preliminary studies established that T. cruzi can be detected by the PCR in the presence of 90% T. rangeli. One hundred thirty-five vectors were collected, and samples were obtained from the rectum, intestines, and stomach and analyzed by microscopy and the PCR. For Triatoma dimidiata rectal samples, the PCR sensitivity (39.1% T. cruzi positive) and the microscopic sensitivity (24.6% positive) was not significantly different. However, in R. prolixus, the PCR proved significantly more sensitive than microscopy: 57.6% positive by PCR compared with 22.7% by microscopy. Rectal samples showed the highest rates of infection followed by intestine and stomach samples. However, 10.5% of the Rhodnius infections would have been missed if only the rectal sample had been analyzed. Thus, the PCR is significantly more sensitive than microscopy for detection of T. cruzi in R. prolixus. Analysis of anatomic sites in addition to the rectal sample may be necessary for accurate assessment of infection in particular vectors. (+info)
Kinetic analysis on nitric oxide binding of recombinant Prolixin-S, a nitric oxide transport protein from the bloodsucking bug, Rhodnius prolixus.
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Kinetics of the NO binding and removal reaction of recombinant Prolixin-S (rProlixin-S) were analyzed using stopped-flow spectrophotometry. The reaction was observed as a biphasic process. The rate constant of the fast phase increased linearly as NO concentration increased. The rate constant at the slow phase increased as NO concentrations increased at low NO concentration, then reached a plateau at high NO concentration. These NO dependencies of the reaction are characteristic of a bimolecular two-step consecutive reaction. The reaction consisted of the fast NO binding reaction of rProlixin-S and the following slow structural change of NO-protein complex. Kinetic studies revealed that the NO binding rate constant was independent of pH, but the rate constant of the NO removal reaction increased as pH increased. The apparent NO dissociation constant (Kd) of rProlixin-S was also calculated from the values of the kinetic parameters obtained in this work. The Kd value increased as pH and temperature increased. The Kd value of rProlixin-S and NO was 10-300 nM in regular physiological condition, which is 103 higher and 103 lower than those of the other ferric and ferrous hemoproteins and NO, respectively. These results indicate that Prolixin-S is one of NO transport proteins regulating blood pressure. (+info)
The distribution of a CRF-like diuretic peptide in the blood-feeding bug Rhodnius prolixus.
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The blood-feeding bug Rhodnius prolixus ingests a large blood meal, and this is followed by a rapid diuresis to eliminate excess water and salt. Previous studies have demonstrated that serotonin and an unidentified peptide act as diuretic factors. In other insects, members of the corticotropin-releasing factor (CRF)-related peptide family have been shown to play a role in post-feeding diuresis. Using fluorescence immunohistochemistry and immunogold labelling with antibodies to the Locusta CRF-like diuretic hormone (Locusta-DH) and serotonin, we have mapped the distribution of neurones displaying these phenotypes in R. prolixus. Strong Locusta-DH-like immunoreactivity was found in numerous neurones of the central nervous system (CNS) and, in particular, in medial neurosecretory cells of the brain and in posterior lateral neurosecretory cells of the mesothoracic ganglionic mass (MTGM). Positively stained neurohaemal areas were found associated with the corpus cardiacum (CC) and on abdominal nerves 1 and 2. In addition, Locusta-DH-like immunoreactive nerve processes were found over the posterior midgut and hindgut. Double-labelling studies for Locusta-DH-like and serotonin-like immunoreactivity demonstrated some co-localisation in the CNS; however, no co-localisation was found in the medial neurosecretory cells of the brain, the posterior lateral neurosecretory cells of the MTGM or neurohaemal areas. To confirm the presence of a diuretic factor in the CC and abdominal nerves, extracts were tested in Malpighian tubule secretion assays and cyclic AMP assays. Extracts of the CC and abdominal nerves caused an increase in the rate of secretion and an increase in the level of cyclic AMP in the Malpighian tubules of fifth-instar R. prolixus. The presence of the peptide in neurohaemal terminals of the CC and abdominal nerves that are distinct from serotonin-containing terminals indicates that the peptide is capable of being released into the haemolymph and that this release can be independent of the release of serotonin. (+info)
A missing metabolic pathway in the cattle tick Boophilus microplus.
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Heme proteins are involved in a wide variety of biological reactions, including respiration, oxygen transport and oxygen metabolism [1]. The heme prosthetic group is synthesized in almost all living organisms except for a few pathogenic bacteria and trypanosomatids that use blood as food [2] [3]. There is a general belief that all nucleated animal cells synthesize heme [1] [4]. However, blood-feeding arthropods ingest enormous amounts of vertebrate blood in a single meal and the heme pathway has not been studied in these animals. We have examined heme synthesis in two hematophagous arthropods - the blood-sucking bug Rhodnius prolixus and the cattle tick Boophilus microplus. We show that R. prolixus makes heme and has a fully operative heme biosynthetic pathway, while B. microplus does not. To our knowledge, this is the first report of an animal that does not synthesize its own heme and relies solely on the recovery of heme present in the diet. Because of the inability of Boophilus to synthesize heme and its ability to deal efficiently with large amounts of free heme, we propose this organism as a good model for studying heme transport and reutilization in animal cells. (+info)
Effects of environmental temperature on life tables of Rhodnius neivai Lent, 1953 (Hemiptera: Reduviidae) under experimental conditions.
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Changes in life tables of Rhodnius neivai due to variations of environmental temperature were studied, based on nine cohorts. Three cohorts were kept at 22 degrees C, three at 27 degrees C and three at 32 degrees C. Cohorts were censused daily during nymphal instars and weekly in adults. Nine complete horizontal life tables were built. A high negative correlation between temperature and age at first laying was registered (r=-0,84). Age at maximum reproduction was significantly lower at 32 degrees C. Average number of eggs/female/week and total eggs/female on its life time were significantly lower at 22 degrees C. Total number of egg by cohort and total number of reproductive weeks were significantly higher at 27 degrees C. At 32 degrees C, generational time was significantly lower. At 27 degrees C net reproductive rate and total reproductive value were significantly higher. At 22 degrees C, intrinsic growth, finite growth and finite birth rates were significantly lower. At 22 degrees C, death instantaneous rate was significantly higher. (+info)
Metalloproteases in Trypanosoma rangeli-infected Rhodnius prolixus.
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Protease activities in the haemolymph and fat body in a bloodsucking insect, Rhodnius prolixus, infected with Trypanosoma rangeli, were investigated. After SDS-polyacrylamide gel electrophoresis containing gelatin as substrate, analysis of zymograms performed on samples of different tissues of controls and insects inoculated or orally infected with short or long epimastigotes of T. rangeli, demonstrated distinct patterns of protease activities: (i) proteases were detected in the haemolymph of insects which were fed on, or inoculated with, short epimastigotes of T. rangeli (39 kDa and 33 kDa, respectively), but they were not observed in the fat body taken from these insects; (ii) protease was also presented in the fat bodies derived from naive insects or controls inoculated with sterile phosphate-saline buffer (49 kDa), but it was not detected in the haemolymph of these insects; (iii) no protease activity was observed in both haemolymph and fat bodies taken from insects inoculated with, or fed on, long epimastigotes of T. rangeli. Furthermore, in short epimastigotes of T. rangeli extracts, three bands of the protease activities with apparent molecular weights of 297, 198 and 95 kDa were detected while long epimastigotes preparation presented only two bands of protease activities with molecular weights of 297 and 198 kDa. The proteases from the insect infected with T. rangeli and controls belong to the class of either metalloproteases or metal-activated enzymes since they are inhibited by 1,10-phenanthroline. The significance of these proteases in the insects infected with short epimastigotes of T. rangeli is discussed in relation to the success of the establishment of infection of these parasites in its vector, R. prolixus. (+info)