Role of host lysosomal associated membrane protein (LAMP) in Trypanosoma cruzi invasion and intracellular development

Trypanosoma cruzi host cell entry depends on lysosomes for the formation of the parasitophorous vacuole. Lysosome internal surface is covered by two major proteins, highly sialilated, Lysosome Associated Membrane Proteins 1 and 2. T. cruzi, on the other hand, needs to acquire sialic acid from its host cell through the activity of trans-sialidase, an event that contributes to host cell invasion and later for parasite vacuole escape. Using LAMP1/2 knock out cells we were able to show that these two proteins are important for T. cruzi infection of host cells, both in entrance and intracellular development, conceivably by being the major source of sialic acid for T. cruzi.     

Coinfection with different Trypanosoma cruzi strains interferes with the host immune response to infection

A century after the discovery of Trypanosoma cruzi in a child living in Lassance, Minas Gerais, Brazil in 1909, many uncertainties remain with respect to factors determining the pathogenesis of Chagas disease (CD). Herein, we simultaneously investigate the contribution of both host and parasite factors during acute phase of infection in BALB/c mice infected with the JG and/or CL Brener T. cruzi strains. JG single infected mice presented reduced parasitemia and heart parasitism, no mortality, levels of pro-inflammatory mediators (TNF-α, CCL2, IL-6 and IFN-γ) similar to those found among naïve animals and no clinical manifestations of disease. On the other hand, CL Brener single infected mice presented higher parasitemia and heart parasitism, as well as an increased systemic release of pro-inflammatory mediators and higher mortality probably due to a toxic shock-like systemic inflammatory response. Interestingly, coinfection with JG and CL Brener strains resulted in intermediate parasitemia, heart parasitism and mortality. This was accompanied by an increase in the systemic release of IL-10 with a parallel increase in the number of MAC-3⁺ and CD4⁺ T spleen cells expressing IL-10. Therefore, the endogenous production of IL-10 elicited by coinfection seems to be crucial to counterregulate the potentially lethal effects triggered by systemic release of pro-inflammatory mediators induced by CL Brener single infection. In conclusion, our results suggest that the composition of the infecting parasite population plays a role in the host response to T. cruzi in determining the severity of the disease in experimentally infected BALB/c mice. The combination of JG and CL Brener was able to trigger both protective inflammatory immunity and regulatory immune mechanisms that attenuate damage caused by inflammation and disease severity in BALB/c mice.     

Focusing of pepsin in strongly acidic immobilized pH gradients

A new acrylamido buffer has been synthesized, for use in isoelectric focusing in immobilized pH gradients. This compound (2-acrylamido glycolic acid) has a pK = 3.1 (at 25 degrees C, 20 mM concentration during titration) and is used, by titration with the pK 9.3 Immobiline, to produce a linear pH gradient in the pH 2.5-3.5 interval. Pepsin (from pig stomach) focused in this acidic pH gradient is resolved into four components, two major (with pI values 2.76 and 2.78) and two minor (having pI values 2.89 and 2.90). This is the first time that such strongly acidic proteins could be focused in an immobilized pH gradient. Even in conventional isoelectric focusing in amphoteric buffers it has been impossible to focus reproducibly very-low-pI macromolecules.     

Physico-chemical properties of amphoteric, isoelectric, macroreticulate buffers

We report here the properties of a new family of resins possessing an amphoteric character and able to strongly buffer at their pI values. They have been adopted as carriers for growth of cells in tissue culture and for hydroponics (Righetti et al. 1991; J. Biotechnol. 17, 169-176) but it is to be expected that such resins could have interesting chromatographic applications. It has been found that such beads [made by incorporating a pK 6.2 weak acrylamido base and a pK 4.6 weak acrylamido acid in a 2:1 ratio (thus with a pI of 6.2) into a neutral polyacrylamide backbone], independently from their initial conditioning (acid- or base-washed), spontaneously seek their equilibrium position (pI value) upon washing off excess titrant. Thus, upon potentiometric titration, they are seen to buffer in both directions of the pH scale (contrary to the behaviour of a pure carboxyl or a pure amino surface, which will exhibit only unidirectional buffering power). From the behaviour of these amphoteric beads when polymerized in the absence or in the presence of salts (0.2 M NaCl), it is hypothesized that, for exerting buffering power, both the buffering ion and its counterion must be incorporated non-randomly in the chain, but as a couple or in close proximity. Upon random incorporation of the two ions, buffering power is lost.     

Cell culture and animal infection with distinct Trypanosoma cruzi strains expressing red and green fluorescent proteins

Different strains of Trypanosoma cruzi were transfected with an expression vector that allows the integration of green fluorescent protein (GFP) and red fluorescent protein (RFP) genes into the beta-tubulin locus by homologous recombination. The sites of integration of the GFP and RFP markers were determined by pulse-field gel electrophoresis and Southern blot analyses. Cloned cell lines selected from transfected epimastigote populations maintained high levels of fluorescent protein expression even after 6 months of in vitro culture of epimastigotes in the absence of drug selection. Fluorescent trypomastigotes and amastigotes were observed within Vero cells in culture as well as in hearts and diaphragms of infected mice. The infectivity of the GFP- and RFP-expressing parasites in tissue culture cells was comparable to wild type populations. Furthermore, GFP- and RFP-expressing parasites were able to produce similar levels of parasitemia in mice compared with wild type parasites. Cell cultures infected simultaneously with two cloned cell lines from the same parasite strain, each one expressing a distinct fluorescent marker, showed that at least two different parasites are able to infect the same cell. Double-infected cells were also detected when GFP- and RFP-expressing parasites were derived from strains belonging to two distinct T. cruzi lineages. These results show the usefulness of parasites expressing GFP and RFP for the study of various aspects of T. cruzi infection including the mechanisms of cell invasion, genetic exchange among parasites and the differential tissue distribution in animal models of Chagas disease.     

Ingestion and incorporation of freshwater diatoms by Daphnia pulicaria: do morphology and oxylipin production matter?

In order to explain differences in the growth and reproductionof Daphnia pulicaria fed various freshwater diatoms, we measuredingestion rates and carbon incorporation for six cultured diatomspecies: the single-celled Stephanodiscus hantzschii, Stephanodiscusminutulus and Cyclotella meneghiniana, and the colony-formingAsterionella formosa, Fragilaria capucina and Fragilaria sp.Two of the colony-forming species, when damaged, produced polyunsaturatedaldehydes (oxylipins) that have been found to impair the reproductionof marine copepods. We tested two hypotheses: (i) feeding andincorporation rates are affected by diatom morphology; and (ii)polyunsaturated aldehydes act as feeding deterrents. Daphniabody length versus ingestion rate regressions differed for single-celledand colony-forming diatoms. Ingestion rates for single-celleddiatoms showed clear size dependencies and high correlationcoefficients, while the dependency was weak for colony-formingdiatoms and individual variability was high. This differencewas not observed for carbon incorporation rates, which showedlow variability for all diatoms. Asterionella formosa yieldedthe lowest incorporation rates due to low incorporation efficiency,while all other diatoms were incorporated at similar rates.Thus, morphological differences of the diatoms had no effecton carbon uptake by Daphnia. The presence or absence of polyunsaturatedaldehydes did not cause different ingestion rates; hence thealdehydes are not feeding deterrents.     

Mitochondrial evidence for distinct phylogeographic units in the endangered Malagasy poison frog Mantella bernhardi

Mantella bernhardi is an endemic species of Malagasy poison frog threatened by loss and fragmentation of its natural habitat and collection for the pet trade. It is classified as threatened according to the International Union for Conservation of Nature and Natural Resources (IUCN) categories and included in Appendix II of the Convention on the International Trade of Endangered Species (CITES). A recent survey has increased the known distributional range of the species from one to eight populations across southeastern Madagascar, but little is known about its biology and genetic diversity. Here we estimate inter- and intrapopulation mitochondrial genetic variation of four populations. Populations from the northern and southern parts of the distributional range showed a high degree of divergence (maximum of 11.35% in cytochrome b) and were recovered as reciprocally monophyletic groups. Nine haplotypes were detected in the northern and 12 in the southern populations. The population from Ranomafana National Park showed the lowest number of haplotypes and nucleotide diversity, and shared its most common haplotype with the second northern population from Tolongoina. All the other detected haplotypes were unique to each of the four populations. This suggests the existence of important barriers to gene flow, pre-dating human colonization of Madagascar at about 2000 years ago, in distinct contrast to other Mantella species that show a high degree of haplotype sharing throughout their range. The continued habitat fragmentation within the distribution range of M. bernhardi prevents any connection between its populations. Our data indicate the existence of at least two different management units for conservation in this species, corresponding to the North and South of its distribution range, and highlight the existence of strong regional endemism in southeastern Madagascar.     

Blockade of endothelin ET(A)/ET(B) receptors favors a role for endothelin during acute Trypanosoma cruzi infection in rats

Endothelin has been implicated in the pathogenesis of experimental and human Chagas' disease (American trypanosomiasis). In the present study, we tested the effect of bosentan, an antagonist of both ET(A) and ET(B) endothelin receptors, on parasitemia, histopathology (heart and diaphragm), heart levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, interferon (IFN)-gamma, CCL2, CCL3 and CCL5, and the serum levels of nitrate/nitrite (NOx). Bosentan treatment was accompanied by a significant increase in parasitemia and tissue parasitism or inflammation. In vehicle-treated rats, Trypanosoma cruzi infection increased the cardiac levels of TNF-alpha, IFN-gamma and IL-10, at day 9 post inoculation, and the TNF-alpha remained elevated until day 13. The infection also caused a significant increase in the cardiac levels of the chemokines CCL2 (9, 13 and 18 days) and CCL3 (13 and 18 days). Bosentan-treatment had no significant effect on the infection-associated increase in IFN-gamma and chemokine concentrations. There was a lower increase in IL-10 at day 9 and this was mirrored by a greater increase of TNF-alpha at day 13, in comparison with vehicle-treated rats. These latter findings correlated well with the enhanced inflammatory process in hearts of bosentan-treated infected rats. Bosentan treatment reduced the infection-associated increase in NOx serum concentration. Altogether, our data suggest that ET action on ET(A) and ET(B) receptors may play a role in the initial control of T. cruzi infection in rats probably by interfering in NO production.     

On the Possible Role of tRNA Base Modifications in the Evolution of Codon Usage: Queuosine and Drosophila

The best documented selection-based hypothesis to explain unequal usage of codons is based on the relative abundance of isoaccepting tRNAs. In unicellular organisms the most used codons are optimally translated by the most abundant tRNAs. The chemical bonding energies are affected by modification of the four traditional bases, in particular in the first anti-codon corresponding to the third codon position. One nearly universal modification is queuosine (Q) for guanine (G) in tRNA^His, tRNA^Asp, tRNA^Asn, and tRNA^Tyr; this changes the optimal binding from codons ending in C to no preference or a slight preference for U-ending codons. Among species of Drosophila, codon usage is constant with the exception of the Drosophila willistoni lineage which has shifted primary usage from C-ending codons to U/T ending codons only for these four amino acids. In Drosophila melanogaster Q containing tRNAs only predominate in old adults. We asked the question whether in D. willistoni these Q containing tRNAs might predominate earlier in development. As a surrogate for levels of modification we studied the expression of the gene (tgt) coding for the enzyme that catalyzes the substitution of Q for G in different life stages of D. melanogaster, D. pseudoobscura, and D. willistoni. Unlike the other two species, the highest tgt expression in D. willistoni is in young females producing eggs. Because tRNAs laid down in eggs persist through the early stages of development, this implies that Q modification occurs earlier in development in D. willistoni than in other Drosophila.     

Meristem culture and virus eradication in Alstroemeria

Stem apical meristems, rhizome apical meristems and rhizome axillary meristems excised from Alstroemeria plants were grown in vitro on modified Murashige and Skoog (MS) media containing different concentrations of gibberellic acid and 6-benzylaminopurine (BA). Plantlets developed from stem apical meristems never regenerated a rhizome and eventually died. The highest regeneration rate (74.1%) of plantlets with a rhizome was observed when rhizome axillary meristems were grown on modified MS medium containing M 8.9 of BA. Alstroemeria mosaic potyvirus (AlMV) could be eradicated from infected Alstroemeria plants through meristem culture. The rate of virus eradication was 73.7 and 14.7% for plantlets developing from explants measuring 0.7 mm and 2.0 mm, respectively. Greenhouse evaluation of virus-negative and AlMV-infected Alstroemeria plants showed that healthy plants produced more floral stems, more vegetative stems, longer floral stems and gave a higher fresh weight than infected plants.