Internalization of components of the host cell plasma membrane during infection by Trypanosoma cruzi

Epimastigote and trypomastigote forms of Trypanosoma cruzi attach to the macrophage surface and are internalized with the formation of a membrane bounded vacuole, known as the parasitophorous vacuole (PV). In order to determine if components of the host cell membrane are internalized during formation of the PV we labeled the macrophage surface with fluorescent probes for proteins, lipids and sialic acid residues and then allowed the labeled cells to interact with the parasites. The interaction process was interrupted after 1 hr at 37ºC and the distribution of the probes analyzed by confocal laser scanning microscopy. During attachment of the parasites to the macrophage surface an intense labeling of the attachment regions was observed. Subsequently labeling of the membrane lining the parasitophorous vacuole containing epimastigote and trypomastigote forms was seen. Labeling was not uniform, with regions of intense and light or no labeling. The results obtained show that host cell membrane lipids, proteins and sialoglycoconjugates contribute to the formation of the membrane lining the PV containing epimastigote and trypomastigote T. cruzi forms. Lysosomes of the host cell may participate in the process of PV membrane formation.

Isolation and characterization of the full-length cDNA encoding a member of a novel cytochrome p450 family (CYP320A1) from the tropical freshwater snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni

Cytochrome p450s (cyp450s) are a family of structurally related proteins, with diverse functions, including steroid synthesis and breakdown of toxins. This paper reports the full-length sequence of a novel cyp450 gene, the first to be isolated from the tropical freshwater snail Biomphalaria glabrata, an important intermediate host of Schistosoma mansoni. The nucleotide sequence is 2291 bp with a predicted amino acid sequence of 584aa. The sequence demonstrates conserved cyp450 structural motifs, but is sufficiently different from previously reported cyp450 sequences to be given a new classification, CYP320A1. Initially identified as down-regulated in partially resistant snails in response to S. mansoni infection, amplification of this gene using RT-PCR in both totally resistant or susceptible snail lines when exposed to infection, and all tissues examined, suggests ubiquitous expression. Characterization of the first cyp450 from B. glabrata is significant in understanding the evolution of these metabolically important proteins.

Oocyst wall formation and composition in coccidian parasites

The oocyst wall of coccidian parasites is a robust structure that is resistant to a variety of environmental and chemical insults. This resilience allows oocysts to survive for long periods, facilitating transmission from host to host. The wall is bilayered and is formed by the sequential release of the contents of two specialized organelles - wall forming body 1 and wall forming body 2 - found in the macrogametocyte stage of Coccidia. The oocyst wall is over 90% protein but few of these proteins have been studied. One group is cysteine-rich and may be presumed to crosslink via disulphide bridges, though this is yet to be investigated. Another group of wall proteins is rich in tyrosine. These proteins, which range in size from 8-31 kDa, are derived from larger precursors of 56 and 82 kDa found in the wall forming bodies. Proteases may catalyze processing of the precursors into tyrosine-rich peptides, which are then oxidatively crosslinked in a reaction catalyzed by peroxidases. In support of this hypothesis, the oocyst wall has high levels of dityrosine bonds. These dityrosine crosslinked proteins may provide a structural matrix for assembly of the oocyst wall and contribute to its resilience.

Compatibility and ectomycorrhiza formation among Pisolithus Isolates and Eucalyptus spp

Twenty-nine isolates of the ectomycorrhiza fungus Pisolithus sp. from different geographical and host origins were tested for their ability to form ectomycorrhizae on Eucalyptus grandis and E. urophylla seedlings under greenhouse conditions. The ectomycorrhiza-forming capacity of isolates varied greatly from one eucalypt species to the other. All isolates from Eucalyptus, nine from Pinus spp. and two isolates from unknown hosts formed mycorrhizae with E. grandis and E. urophylla. Root colonization rates varied from 0 to 5.2 % for all Pinus isolates and those from unknown hosts. Colonization rates for these isolates were lower than those observed for Eucalyptus isolates (0.8 to 89.4 %). Three isolates from unknown hosts formed mycorrhizae with neither Eucalyptus species. The main characteristic for distinguishing Pinus from Eucalyptus isolates was mantle color. These data corroborate previous results obtained in our laboratory indicating that the isolates tested represent at least two distinct different species within the genus Pisolithus.

Total lipid and fatty acid accumulation during basidiospore formation in the ectomycorrhizal fungus Pisolithus sp.

The basidiospores of Pisolithus sp. contain large amounts of lipids, indicating provision for future germination in the host rhizosphere. However, the accumulation, composition, and mobilization of lipids during formation and germination of these spores are largely unknown. In this study, lipid storage and fatty acid composition during basidiosporogenesis were analyzed in fresh basidiocarps using bright-field microscopy and gas chromatography. Abundant lipid bodies are found in the hyphae, basidia, and basidiospores of fungal basidiocarps. This evidences a considerable C transport in the basidiocarp to meet the C demand during basidiospore formation. Fatty acid composition analysis revealed the presence of 24 compounds with chains of 9 to 18 C atoms, either saturated or insaturated, with one or two insaturations. The fatty acid composition and content varied according to the developmental stage of the peridioles. In free basidiospores, the predominant compounds were 16:0, 16:1w5c, 18:1w9c, and 18:2w6,9c/18:0ante, at concentrations of 76, 46, 192, and 51 µg g-1 dry matter, respectively. Our results indicate that oleic acid is the major constituent of lipid reserves in Pisolithus sp. basidiospores. Further studies are being conducted to determine the factors that induce lipid mobilization during spore germination.

In vitro ectomycorrhiza formation by monokaryotic and dikaryotic isolates of Pisolithus microcarpus in Eucalyptus grandis

The formation of ectomycorrhizas by monokaryotic and dikaryotic isolates of Pisolithus microcarpus (Cooke & Massee) G. Cunn. in Eucalyptus grandis W. Hill ex Maid. was studied by in vitro synthesis in Petri dishes. The formation of ectomycorrhizas was observed for all strains tested. Ectomycorrhizas formed by the monokaryotic strains presented a sheath of hyphae around the roots and a Hartig net limited to the epidermis layer, typical of the angiosperm ectomycorrhizas. Colonization rates, a measure of the number of ectomycorrhizas in relation to the total number of lateral root tips, varied from 23 to 62%. Some monokaryotic strains stimulated the formation of lateral roots, promoting increases of up to 109% above the control. The presence of some of the isolates in the in vitro synthesis medium stimulated the production of thicker lateral root tips. The dimensions of the lateral roots tips and ectomycorrhizas varied from one isolate to the next, indicating a variation in their capacity to provoke morphological changes in the host plant roots. The dikaryotic strain M5M11 presented higher values for lateral root yield, number of ectomycorrhizas, and colonization percentage than the corresponding monokaryotic strains, M5 and M11. This indicated the possibility of selecting compatible performing monokaryotic isolates for the yield of superior dikaryotic strains. The set of monokaryotic strains tested varied greatly in their ability to colonize E. grandis roots and cause secondary metabolism-related morphological changes in roots, providing a wealth of model systems for the study of genetic, physiological, and morphogenetic processes involved in Pisolithus-Eucalyptus ectomycorrhiza formation.

The use of confocal laser scanning microscopy to analyze the process of parasitic protozoon-host cell interaction

In this communication we review the results obtained with the confocal laser scanning microscope to characterize the interaction of epimastigote and trypomastigote forms of Trypanosoma cruzi and tachyzoites of Toxoplasma gondii with host cells. Early events of the interaction process were studied by the simultaneous localization of sites of protein phosphorylation, revealed by immunocytochemistry, and sites of actin assembly, revealed by the use of labeled phaloidin. The results obtained show that proteins localized in the interaction sites are phosphorylated. The process of formation of the parasitophorous vacuole was monitored by labeling the host cell surface with fluorescent probes for lipids (PKH26), proteins (DTAF) and sialic acid (FITC-thiosemicarbazide) before interaction with the parasites. Evidence was obtained indicating transfer of components of the host cell surface to the parasite surface in the beginning of the interaction process. We also analyzed the distribution of cytoskeletal structures (microtubules and microfilaments visualized with specific antibodies), mitochondria (visualized with rhodamine 123), the Golgi complex (visualized with C6-NBD-ceramide) and the endoplasmic reticulum (visualized with anti-reticulin antibodies and DIOC6) during the evolution of intracellular parasitism. The results obtained show that some, but not all, structures change their position during evolution of the intracellular parasitism.

How middle managers contribute to strategy formation process: connection of strategy processes and strategy practices

The role of middle management is essential when managing integrative and emergent strategy formation processes. We stand out the importance of its role connecting micro and macro organizational level offering a very important contribution when examining the strategy-as-practice perspective and integrative strategy formation process. The main goal of this research is to analyse the relationship between the integrative strategy formation process and the roles of middle management under the strategy-as-practice perspective. To check it out we adopted a qualitative methodology droving a case analysis in a Spanish University. Data was collected by means of personal interviews with members of different levels of the Institution, documents analysis and direct observation. In advance of some results we find out that the University develops an integrative strategy formation process and confers to middle management an important role extended all over the organization.

The epidemiology and control of schistosomiasis mansoni where Biomphalaria tenagophila is the snail host

The epidemiology and control of schistosomiasis mansoni in the Municipality of Pedro de Toledo (State of S. Paulo, Brazil) since 1980, has been studied. In 1980 the prevalence evaluated by stool exams (Kato-Katz method) was 22.8% and no statistical difference at 5.0% level was observed between rural and urban zones. The intensity of infection was low (58.5 eggs/g of faeces); the highest prevalence and intensity of infection rates were observed within the group of from 5 to 29 years of age, respectively. The transmission of schistosomiasis usually occurred during leisure time. The majority of the carriers of the parasite were asymptomatic. Of the B. tenagophila examined only 0.4% were found to be infected. The control programme has been intensified from 1981 on resulting in a sharp decrease in the prevalence from 22.8% in 1980 to 6% at the present time. This result shows that, in spite of the control programme there is a residual human prevalence. A beginning has been made on the investigation into the possible causes of this residual prevalence (6.0% was maintained through out 1987).

Studies on mosquitoes (Diptera: Culicidae) and anthropic environment. 12- Host-seeking behaviour of Anopheles albitarsis s.l. in South-Eastern Brazil

Results obtained with Anopheles albitarsis s.l. catches mainly performed through human bait at the Ribeira Valley region, SP (Brazil), are reported. Two species of the complex were recognized, namely An. albitarsis s.s. and species B. This latter predominated both in the rice fields and in the dwelling environments. The crepuscular rhythms showed an unimodal sunset pattern with most blood-seeking females caught during dusk. The absence of differences between indoor and outdoor behavior was confirmed for both species of the complex.

Persisting problems related to race and ethnicity in public health and epidemiology research

A recent and comprehensive review of the use of race and ethnicity in research that address health disparities in epidemiology and public health is provided. First it is described the theoretical basis upon which race and ethnicity differ drawing from previous work in anthropology, social science and public health. Second, it is presented a review of 280 articles published in high impacts factor journals in regards to public health and epidemiology from 2009-2011. An analytical grid enabled the examination of conceptual, theoretical and methodological questions related to the use of both concepts. The majority of articles reviewed were grounded in a theoretical framework and provided interpretations from various models. However, key problems identified include a) a failure from researchers to differentiate between the concepts of race and ethnicity; b) an inappropriate use of racial categories to ascribe ethnicity; c) a lack of transparency in the methods used to assess both concepts; and d) failure to address limits associated with the construction of racial or ethnic taxonomies and their use. In conclusion, future studies examining health disparities should clearly establish the distinction between race and ethnicity, develop theoretically driven research and address specific questions about the relationships between race, ethnicity and health. One argue that one way to think about ethnicity, race and health is to dichotomize research into two sets of questions about the relationship between human diversity and health.

Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

The authors have standardized methods for evaluation of the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The general principle of the first method was based on methemoglobin formation by sodium nitrite followed by stimulation of the glucose-6-phosphate dehydrogenase with methylene blue. Forty six adults (23 males and 23 females) were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. The results showed that methemoglobin reduction by methylene blue was 154.40 and 139.90 mg/min (p<0.05) for males and females, respectively, in whole blood, and 221.10 and 207.85 mg/min (n.s.), respectively, in washed red cells. These data showed that using washed red cells and 0.7g% sodium nitrite concentration produced no differences between sexes and also shortened reading time for the residual amount of methemoglobin to 90 minutes. Glutathione reductase activity was evaluated on the basis of the fact that cystamine (a thiol agent) binds to the SH groups of hemoglobin, forming complexes. These complexes are reversed by the action of glutathione reductase, with methemoglobin reduction occurring simultaneously with this reaction. Thirty two adults (16 males and 16 females) were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. Methemoglobin reduction by cystamine was 81.27 and 91.13 mg/min (p<0.01) for males and females, respectively. These data showed that using washed red cells and 0.1 M cystamine concentration permits a reading of the residual amount of methemoglobin at 180 minutes of incubation. Glutathione reductase activity was evaluated by methemoglobin reduction by cystamine in 14 females before and after treatment with 10 mg riboflavin per day for 8 days. The results were 73.69 and 94.26 jug/min (p<0.01) before and after treatment, showing that riboflavin treatment increase glutathione reductase activity even in normal individuals. Three Black G6PD-deficient individuals (2 males and 1 female) were also studied. The G6PD and glutathione reductase were partially activated, the change being more intense in males. On the basis of race and of the laboratory characteristics observed, it is possible to suggest that the G6PD deficiency of these individuals is of the African type and that the female is heterozygous for this deficiency. Analysis of the results as a whole permitted us to conclude that the methods proposed here were efficient for evaluating the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The latter is dependent on the pentose pathway, which generates NADPH, and on riboflavin, a FAD precursor vitamin.

Schistosomiasis mansoni in the municipality of Pedro de Toledo (São Paulo, Brazil) where the Biomphalaria tenagophila is the snail host: I - Prevalence in human population (

Due to the scarce information about the epidemiological features of schistosomiasis in which the vector is Biomphalaria tenagophila, an investigation was carried in Pedro de Toledo in 1980 where such peculiarity is observed. Stool examinations (Kato-Katz method) were performed in 4,741 individuals (22.8% positive to Schistosoma mansoni eggs) of this 583 had previously received chemoterapy and 4,158 remainders, untreated. The schistosomiasis prevalence in those two groups where respectively 31.7% and 21.6%. Epidemiological investigation showed that 83.6% were autochthonous cases from the studied area: the autochthonous prevalence rate, and the intensity of infection in the untreated autochthonous cases were higher in males than in females; the intensity in the latter untreated group was low, 58.5 eggs/g feces (geometric mean). Moreover, according to the age groups the intensity of infections correlated well (r s = 0.745) with the prevalence rates. Schistosomiasis was verified to occur mostly during the leisure time and by the use of water streams for housework in rural zone. Only 0.4% out of 1,137 snails was positive for S. mansoni cercariae, apparently unchanged from the 1978 study when the human prevalence was 12.0%. The studied area presented differences and similarities in relation to the other Brazilian areas were the main intermediate host is B. glabrata.

Chlamydospore formation by Paracoccidioides brasiliensis mycelial form

To investigate the role of some adverse environmental conditions in chlamy-dospore formation by the mycelial form of P. brasiliensis, we cultured four P. brasiliensis isolates (18, Bt4, 1183, Pb9) at 25°C within solid agar medium either rich or poor in nutrients. Isolates 18 and 1183 were also cultured under anaerobiosis in a nitrogen atmosphere. Isolate 18 produced great number of terminal and intercalary chlamydospore after 7-10 days of culture in a medium poor in nutrients (2% agar with 0.1% dextrose and polypepton). The three other isolates also produced chlamydospores under the same conditions, but in lower numbers. Chlamydospore production by isolate 18 was abolished when the fungus was cultured in two agar media rich in nutrients (brain heart infusion and potato dextrose agar). Anaerobic incubation of isolate 18 under an atmosphere of N2 showed small mycelial outgrowth with numerous chlamydospores. At the electron microscopical level, the chlamydospores showed one or various nuclei and numerous mitochondria, indicating great potential for further development. Accordingly, chlamydospores produced multiple budding after only 24 h incubation at 35°C. The results demonstrate that under adverse environmental conditions P. brasiliensis mycelial form produces chlamydospores within a short period of time.

Cyclophosphamide effect on coccidioidomycosis in the rat

Coccidioidomycosis is a systemic mycosis, endemic in arid areas of the American continent. The rat was employed as an experimental host, since it had been shown to reproduce human lesions and present a chronic course of disease with granulomas mainly restricted to lungs. Given the influence of immunosuppressive therapy on the clinical course of human coccidioidomycosis, we studied the effect of cyclophosphamide (CY) in the experimental rat model. Accordingly, animals were inoculated with 400 Coccidioides immitis arthroconidia of the Acosta strain, by intracardiacal route. As single CY doses failed to alter the course of disease, three schedules were used: A) 4 daily doses of 20 mg/kg each, prior to C. immitis inoculation; B) 4 similar daily doses after infection; and C); 6 doses of 20 mg/kg each, given from day +1 to +4, then on days +8 and +9, post infection (pi), taking day 0 as the time of fungal inoculation. The first two schedules inhibited antibody formation up to day 28 pi, without modifying cellular response to coccidioidin as measured by foodpad swelling. Initially, there was greater fungal spread than in controls receiving C. immitis alone, which proved self-limiting in the latter. In contrast, schedule C led to 559r mortality, with both humoral and cellular response abrogation, accompanied by extensive C. immitis dissemination. Histology disclosed significant alterations, such as the persistence of primary infection sporangia, corresponding to the acute stage of coccidioidomycosis in the absence of granuloma development. Therefore, the observed depression in cellular immunity seems responsible for the lack of inflammatory reaction capable of restricting sporangia proliferation in tissues which, in turn, enhances pathogen spread and mortality rate.