Trypanosoma cruzi infection in the opossum Didelphis marsupialis: absence of neonatal transmission and protection by maternal antibodies in experimental infections

The high rate of natural Trypanosoma cruzi infection found in opossums does not always correlate with appreciable densities of local triatomid populations. One alternative method which might bypass the invertebrate vector is direct transmission from mother to offspring. This possibility was investigated in five T. cruzi infected females and their litters (24 young). The influence of maternal antibodies transferred via lactation, on the course of experimental infection, was also examined. Our results show that neonatal transmission is probably not responsible for the high rate of natural T. cruzi infection among opossums. In addition antibodies of maternal origin confer a partial protection to the young. This was demonstrated by the finding of a double prepatency period and 4,5 fold lower levels of circulating parasites, in experimentally infected pouch young from infected as compared to control uninfected mothes. On the other hand, the duration of patent parasitemia was twice as long as that observed in the control group.

Ghost protein damage by peroxynitrite and its protection by melatonin

We have studied the effect of peroxynitrite (ONOO-) on the membrane cytoskeleton of red blood cells and its protection by melatonin. Analysis of the protein fraction of the preparation by SDS-PAGE revealed a dose-dependent (0-600 µM ONOO-) disappearance at pH 7.4 of the main proteins: spectrin, band 3, and actin, with the concomitant formation of high-molecular weight aggregates resistant to reduction by ß-mercaptoethanol (2%) at room temperature for 20 min. These aggregates were not solubilized by 8 M urea. Incubation of the membrane cytoskeleton with ONOO- was characterized by a marked depletion of free sulfhydryl groups (50% at 250 µM ONOO-). However, a lack of effect of ß-mercaptoethanol suggests that, under our conditions, aggregate formation is not mediated only by sulfhydryl oxidation. The lack of a protective effect of the metal chelator diethylenetriaminepentaacetic acid confirmed that ONOO--induced oxidative damage does not occur only by a transition metal-dependent mechanism. However, we demonstrated a strong protection against cytoskeletal alterations by desferrioxamine, which has been described as a direct scavenger of the protonated form of peroxynitrite. Desferrioxamine (0.5 mM) also inhibited the loss of tryptophan fluorescence observed when the ghosts were treated with ONOO-. Glutathione, cysteine, and Trolox® (1 mM), but not mannitol (100 mM), were able to protect the proteins against the effect of ONOO- in a dose-dependent manner. Melatonin (0-1 mM) was especially efficient in reducing the loss of spectrin proteins when treated with ONOO- (90% at 500 µM melatonin). Our findings show that the cytoskeleton, and in particular spectrin, is a sensitive target for ONOO-. Specific antioxidants can protect against such alterations, which could seriously impair cell dynamics and generate morphological changes.

Reduced protective effect of Plasmodium berghei immunization by concurrent Schistosoma mansoni infection

Studies on concomitant schistosomiasis and human and experimental malaria have shown a variation in the immunospecific response, as well as an increase in the severity of both parasitoses. In the present study, a murine co-infection model was used to determine the effects of a co-infection with Schistosoma mansoni and Plasmodium berghei on the protective immunity acquired by repeated malarial infections and subsequent curative treatment with chloroquine. Our results have demonstrated that, compared to an infection with P. berghei only, the co-infection increases the malarial parasitaemia and decreases the survival rate. Indeed, mice that were immunized by infection and treatment with drug displayed no mortality whereas co-infected mice showed a reduced protective efficacy of immunization against P. berghei (mortality > 60%). Interestingly, this high mortality rate was not associated with high levels of parasitaemia. Our findings support the idea of a suppressive effect of a Schistosoma co-infection on the anti-malarial protection by immunization. This result reveals a possible drawback of the development of anti-malarial vaccines, especially considering the wide endemic areas for both parasitoses.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80ºC) and under high pressure conditions at low temperature (3.75 kbar, -13ºC). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR) method

We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

Duffy blood group genotypes among malaria patients in Rondônia, Western Brazilian Amazon

We have compared Duffy blood group genotype distribution, as determined by polymerase chain reaction with allele-specific primers, in 68 Plasmodium vivax-infected patients and 59 non-vivax malaria controls from Rondônia, Brazil. Homozygosity for the allele Fy, which abolishes Duffy antigen expression on erythrocytes, was observed in 12% non-vivax controls but in no P. vivax patient. However, no significant association was found between Fy heterozygosity and protection against P. vivax. The Fy x allele, which has recently been associated with very weak erythrocyte expression of Duffy antigen, was not found in local P. vivax patients.

Frugivory by birds on Schefflera morototoni (Araliaceae) in a Cerrado-Amazon orest transition area, eastern Mato Grosso, Brazil

Schefflera morototoni fruits are important food source for neotropical frugivorous birds. The objectives of the present study were to record bird species that consumed fruits of S. morototoni in a forest in the transition Cerrado-Amazon Forest, Mato Grosso, Brazil and evaluate the potential of these bird species as seed dispersers of this plant species. During 31 observation hours, from November 1 to 5, 2011, 23 bird species were recorded consuming S. morototoni fruits. Out of these, 20 bird species were considered potential seed dispersers, as they swallow the fruits whole. The species consuming the greatest number of fruits were Aburria cujubi (24% of total consumed fruits), Pteroglossus castanotis (18%), Tangara palmarum (12%), Patagioenas speciosa (11%), Ramphastos toco (8%), and Dacnis lineata (5%). The species T. palmarum showed the highest visit frequency (VF = 1.51), followed by P. castanotis (VF = 0.80), and D. lineata (VF = 0.77). All bird species employed picking foraging method for removal of fruits and in 11 species (48%) this method was the only one used. Agonistic interactions represented 13% of the total number of visits. Dacnis lineata received the highest number of attacks and P. castanotis and Pitangus sulphuratus were the more aggressive species. The high bird richness and the great number of consumed fruits indicated that the fruits of S. morototoni may be an important food resource for birds in the Cerrado-Amazon Forest transition

Use of tree species by White-throated treerunner (Pygarrhichas albogularis King) in a secondary native forest of southern Chile

ABSTRACT In forest ecosystems, numerous species of insectivorous birds use certain tree species as feeding and nesting substrates. Between 2009 and 2010, the use of different floristic components as feeding substrate by the Pygarrhichas albogularis King, 1831 was evaluated in a southern Chilean secondary native forest. From a total of 13 trees and bush species, six tree species were used by P. albogularis as a feeding substrate. Tree use was limited to intermediate heights (11-20 m) and, mainly, to the trunk (40% of observations) and secondary branches (26%). Pygarrhichas albogularis showed a disproportionated use of N. dombeyi and an important use of trees with a greater age structure (DBH 81-100 cm). Nothofagus dombeyi presented a significantly greater tree bark crevice depth than E. cordifolia. In turn, covariance between crevice depth and invertebrate supply in tree bark was positive and significant. We consider bark depth and invertebrate supply to be the proximate causes explaining P. albogularis disproportionated use of Nothofagus dombeyi.

Induction of apoptosis in A549 pulmonary cells by two Paracoccidioides brasiliensis samples

Paracoccidioidomycosis presents a variety of clinical manifestations and Paracoccidioides brasiliensis can reach many tissues, most importantly the lungs. The ability of the pathogen to interact with host surface structures is essential to its virulence. The interaction between P. brasiliensis and epithelial cells has been studied, with particular emphasis on the induction of apoptosis. To investigate the expression of different apoptosis-inducing pathways in human A549 cells, we infected these cells with P. brasiliensis Pb18SP (subcultured) and 18R (recently isolated from cell culture and showing a high adhesion pattern) samples in vitro. The expressions of Bcl-2, Bak and caspase 3 were analysed by flow cytometry and DNA fragmentation using the TUNEL technique. Apoptosis of human A549 cells was induced by P. brasiliensis in a sample and time-dependent manner. Using an in vitro model, our data demonstrates that caspase 3, Bak, Bcl-2 and DNA fragmentation mediate P. brasiliensis-induced apoptosis in A549 cells. The overall mechanism is a complex process, which may involve several signal transduction pathways. These findings could partially explain the efficient behaviour of this fungus in promoting tissue infection and/or blood dissemination.

Nitric oxide production by Peromyscus yucatanicus (Rodentia) infected with Leishmania (Leishmania) mexicana

Peromyscus yucatanicus (Rodentia: Cricetidae) is a primary reservoir of Leishmania (Leishmania) mexicana (Kinetoplastida: Trypanosomatidae). Nitric oxide (NO) generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L.) mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 10 2 and 2.5 x 10 6 promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001) in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L.) mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection.

Mycorrhizal effectiveness on physic nut as influenced by phosphate fertilization levels

In recent years, physic nut (Jatropha curcas L.) has attracted attention because of its potential for biofuel production. Although it is adapted to low-fertility soils, physic nut requires soil acidity corrections and addition of a considerable amount of fertilizer for high productivity. The objective of this research was to evaluate the effectiveness of arbuscular mycorrhizal fungi (AMF) (control without AMF inoculation, Gigaspora margarita inoculation or Glomus clarum inoculation) on increasing growth and yield of physic nut seedlings under different rates of P fertilization (0, 25, 50, 100, 200, and 400 mg kg-1 P soil) in greenhouse. The experiment was arranged in a completely randomized, block in a factorial scheme design with four replications. The physic nut plants were harvested 180 days after the beginning of the experiment. Mycorrhizal inoculation increased physic nut growth, plant P concentration and root P uptake efficiency at low soil P concentrations. The P use quotient of the plants decreased as the amount of P applied increased, and the P use efficiency index increased at low P levels and decreased at high P levels. Mycorrhizal root colonization and AMF sporulation were negatively affected by P addition. The highest mycorrhizal efficiency was observed when the soil contained between 7.8 and 25 mgkg-1 of P. The physic nut plants responded strongly to P application, independent of mycorrhizal inoculation.

UVA I-protection effectiveness of bioactive compound and organic UV filters: an in vitro assessment

This research work aimed at determining the UVA effectiveness (UVA I/UV ratio), by diffuse transmittance analysis, of sunscreens developed with a bioactive substance, the rutin, associating or not with organic UVB-UVA filters incorporated at a phosphate-base O/W emulsion. Sunscreens provided conflicting and unpredictable results concerning the anti-UVA protection, specially, at the UVA I region. Possible interactions among the organic UV filters and the polyphenolic bioactive substance may have accounted with improvement or reduction of UV protection by a complex and not yet elucidated mechanism, probably regarding wavelength delocalization to superior or inferior values, by resonant molecule stabilization or destabilization.

Glicoproteína-P, resistência a múltiplas drogas (MDR) e relação estrutura-atividade de moduladores

Multidrug resistance, MDR is a major obstacle for cancer chemotherapy. MDR can be reversed by drugs that vary in their chemical structure and main biological activity. Many efforts have been done to overcome MDR based on studies of structure-activity relationships and in this review we summarize some aspects of MDR mediated by P-glycoprotein (P-gp), as the most experimentally and clinically tested form of drug resistance. The most significant MDR mechanisms revealed until now are shortly discussed. Physicochemical and structural properties of MDR modulators, measures of the MDR reversal, and QSAR studies are included.

Inheritance of anthracnose resistance in common bean genotypes P.I. 207262 and AB 136

Bean (Phaseolus vulgaris) lines P.I. 207262 and AB 136, both resistant to delta and kappa races of Colletotrichum lindemuthianum, were crossed with Michelite, Dark Red Kidney, and Perry Marrow, susceptible to both races, and with Cornell 49-242, resistant to delta and susceptible to kappa. F1 and F2 reactions demonstrated that P.I. 207262 carries duplicate dominant genes for resistance to the delta race; AB 136 carries a dominant gene. These resistance genes are independent of the Are gene from Cornell 49-242. With respect to the kappa race, F1 and F2 data showed that the resistance controlled by P.I. 207262 and by AB 136 depends on a single dominant gene. Complementary factors were involved with AB 136 resistance to the delta race and with P.I. 207262 resistance to kappa.

Comparison of the diagnosis of malaria by microscopy, immunochromatography and PCR in endemic areas of Venezuela

Whole blood samples (N = 295) were obtained from different locations in Amazonas and Sucre States, in Venezuela. Malaria was diagnosed by microscopy, OptiMAL™ and polymerase chain reaction (PCR), with Plasmodium vivax, P. falciparum, and P. malariae being detected when possible. We identified 93 infections, 66 of which were caused by P. vivax, 26 by P. falciparum, and 1 was a mixed infection. No infection caused by P. malariae was detected. The sensitivity and specificity of each diagnostic method were high: 95.7 and 97.9% for microscopy, 87.0 and 97.9% for OptiMAL, and 98.0 and 100% for PCR, respectively. Most samples (72.2%) showed more than 5000 parasites/µL blood. The sensitivity of the diagnosis by microscopy and OptiMAL decreased with lower parasitemia. All samples showing disagreement among the methods were reevaluated, but the first result was used for the calculations. Parasites were detected in the 6 false-negative samples by microscopy after the second examination. The mixed infection was only detected by PCR, while the other methods diagnosed it as P. falciparum (microscopy) or P. vivax (OptiMAL) infection. Most of the false results obtained with the OptiMAL strip were related to the P. falciparum-specific band, including 3 species misdiagnoses, which could be related to the test itself or to genetic variation of the Venezuelan strains. The use of the microscopic method for malaria detection is recommended for its low cost but is very difficult to implement in large scale, population-based studies; thus, we report here more efficient methods suitable for this purpose.