Lectins from seeds of jack fruit (Artocarpus integrifolia L.): isolation and purification of two isolectins from the albumin fraction

Two isolectins were isolated from the albumin fraction ofArtocarpus integrifolia L. seeds, by precipitation with ammonium sulfate, and DEAE-cellulose and SP-Sephadex C-50 chromatography. The isolectins, when passed through Sephadex G-100 at pH 7.4, had a molecular mass of 43 000 and when subjected to SDS electrophoresis in the presence of β-mercaptoethanol consisted of two subunits with molecular mass of 11 250 and 15 000. When they were tested with human erythrocytes of all the groups of the ABO system there was no blood specificity. The isolectins formed interference arcs by Ouchterlony double diffusion in agarose gel. Part 1.     

Cytochemistry and freeze-fracture of membranes isolated from the electrocyte of Electrophorus electricus (L.)

Summary Membranes were isolated from the main electric organ of Electrophorus electricus and studied by means of cytochemistry and freezefracture. The membrane fractions consisted of vesicles inside-in as determined by localization of anionic sites using colloidal iron and cationized ferritin particles. The anionic sites were not homogeneously distributed on the surface of the vesicle. Freeze-fracture showed the presence of intramembranous particles associated with either protoplasmic (P) or extracellular (E) faces of the membrane. Regions of the membrane without particles were observed. The results are discussed in relation to the existence of association between intramembranous particles and membrane receptors.For all correspondence     

The AMA1-RON complex drives Plasmodium sporozoite invasion in the mosquito and mammalian hosts

Plasmodium sporozoites that are transmitted by blood-feeding female Anopheles mosquitoes invade hepatocytes for an initial round of intracellular replication, leading to the release of merozoites that invade and multiply within red blood cells. Sporozoites and merozoites share a number of proteins that are expressed by both stages, including the Apical Membrane Antigen 1 (AMA1) and the Rhoptry Neck Proteins (RONs). Although AMA1 and RONs are essential for merozoite invasion of erythrocytes during asexual blood stage replication of the parasite, their function in sporozoites was still unclear. Here we show that AMA1 interacts with RONs in mature sporozoites. By using DiCre-mediated conditional gene deletion in P. berghei, we demonstrate that loss of AMA1, RON2 or RON4 in sporozoites impairs colonization of the mosquito salivary glands and invasion of mammalian hepatocytes, without affecting transcellular parasite migration. Three-dimensional electron microscopy data showed that sporozoites enter salivary gland cells through a ring-like structure and by forming a transient vacuole. The absence of a functional AMA1-RON complex led to an altered morphology of the entry junction, associated with epithelial cell damage. Our data establish that AMA1 and RONs facilitate host cell invasion across Plasmodium invasive stages, and suggest that sporozoites use the AMA1-RON complex to efficiently and safely enter the mosquito salivary glands to ensure successful parasite transmission. These results open up the possibility of targeting the AMA1-RON complex for transmission-blocking antimalarial strategies.     

Leishmania braziliensis causing human disease in Northeast Brazil presents loci with genotypes in long-term equilibrium

BackgroundLeishmaniases are neglected tropical diseases that inflict great burden to poor areas of the globe. Intense research has aimed to identify parasite genetic signatures predictive of infection outcomes. Consistency of diagnostic tools based on these markers would greatly benefit from accurate understanding of Leishmania spp. population genetics. We explored two chromosomal loci to characterize a population of L. braziliensis causing human disease in Northeast Brazil.Methodology/Principal findingsTwo temporally distinct samples of L. braziliensis were obtained from patients attending the leishmaniasis clinic at the village of Corte de Pedra: (2008–2011) primary sample, N = 120; (1999–2001) validation sample, N = 35. Parasites were genotyped by Sanger’s sequencing of two 600 base pairs loci starting at nucleotide positions 3,074 and 425,451 of chromosomes 24 and 28, respectively. Genotypes based on haplotypes of biallelic positions in each locus were tested for several population genetic parameters as well as for geographic clustering within the region. Ample geographic overlap of genotypes at the two loci was observed as indicated by non-significant Cusick and Edward’s comparisons. No linkage disequilibrium was detected among combinations of haplotypes for both parasite samples. Homozygous and heterozygous genotypes displayed Hardy-Weinberg equilibrium (HWE) at both loci in the two samples when straight observed and expected counts were compared by Chi-square (p>0.5). However, Bayesian statistics using one million Monte-Carlo randomizations disclosed a less robust HWE for chromosome 24 genotypes, particularly in the primary sample (p = 0.04). Fixation indices (Fst) were consistently lower than 0.05 among individuals of the two samples at both tested loci, and no intra-populational structuralization could be detected using STRUCTURE software.Conclusions/SignificanceThese findings suggest that L. braziliensis can maintain stable populations in foci of human leishmaniasis and are capable of robust genetic recombination possibly due to events of sexual reproduction during the parasite’s lifecycle.     

Distribution dynamics of South American savanna birds in response to Quaternary climate change

Several lines of evidence suggest that savannas currently distributed disjointedly in the southern and northern portions of South America might have been connected and disconnected many times during the Quaternary climatic fluctuations. Here, we investigated how climate change since the Last Interglacial may have modified the distribution of bird species associated with South American savannas. We evaluated the connections between South America's savannas using 10 broadly distributed species and the impact of climate changes in community composition using 18 species endemic to Cerrado. We fit ecological niche models to each of the 28 bird species to compare the potential distribution patterns for the Last Interglacial (120 kyr BP), the Last Glacial Maximum (21 kyr BP) and the present. Our results corroborated hypotheses of past connections between northern and southern blocks of savannas through three hypothetical corridors that existed along the Andes, Atlantic Coast and through central Amazonia. In addition, our results also suggested the existence of a fourth plausible corridor located along the Madeira River, crossing Amazonia from the southwest to the northeast. Finally, our analysis showed significant changes in the community composition dynamics of endemic Cerrado species. Our results further reinforce the notion that climate change has major impacts on the distribution of savanna species.     

Polymorphism in the promoter region of the mannose-binding lectin gene among human T-cell lymphotropic virus infected subjects

The present study investigated the frequency of the mutations at positions -550 and -221 of the mannose-binding lectin (MBL) gene in a sample of 75 human T-cell lymphotropic virus (HTLV) infected patients and 96 HTLV seronegative controls, in order to evaluate the occurrence of a possible association between the polymorphism and HTLV infection. A sequence specific primer-polymerase chain reaction was used for discrimination of the polymorphism. The analysis of allele frequencies at position -550 did not show any significant differences between HTLV infected group and controls, but there was a significant difference at position -221. The comparative analysis of haplotypes frequencies were not significant, but the genotype frequencies between the two groups, revealed a higher prevalence of genotype LYLX (25.3%), associated with medium and low MBL serum levels among HTLV infected subjects. The odds ratio estimation demonstrated that the presence of genotype LYLX was associated with an increased risk of HTLV infection (p = 0.0096; 1.38 < or = IC95% < or = 7.7605). There was no association between proviral load and the promoter polymorphism, but when promoter and exon 1 mutations were matched, it was possible to identify a significant higher proviral load among HTLV infected individuals carrying haplotypes correlated to low serum levels of MBL. The present study shows that the polymorphism in the promoter region of the MBL gene may be a genetic marker associated with HTLV infection, and emphasizes the need for further studies to determinate if the present polymorphism have any impact on diseases linked to HTLV infection.     

Controlled Production of Stable Heterologous Proteins in Lactococcus lactis

The use of Lactococcus lactis (the most extensively characterized lactic acid bacterium) as a delivery organism for heterologous proteins is, in some cases, limited by low production levels and poor-quality products due to surface proteolysis. In this study, we combined in one L. lactis strain use of the nisin-inducible promoter P_nisA and inactivation of the extracellular housekeeping protease HtrA. The ability of the mutant strain, designated htrA-NZ9000, to produce high levels of stable proteins was confirmed by using the staphylococcal nuclease (Nuc) and the following four heterologous proteins fused or not fused to Nuc that were initially unstable in wild-type L. lactis strains: (i) Staphylococcus hyicus lipase, (ii) the bovine rotavirus antigen nonstructural protein 4, (iii) human papillomavirus antigen E7, and (iv) Brucella abortus antigen L7/L12. In all cases, protein degradation was significantly lower in strain htrA-NZ9000, demonstrating the usefulness of this strain for stable heterologous protein production.     

Integrin α8β1 regulates adhesion,migration and proliferation of human intestinal crypt cells via a predominant RhoA/ROCK‐dependent mechanism

Background. Integrins are transmembrane αβ heterodimer receptors that function as structural and functional bridges between the cytoskeleton and ECM (extracellular matrix) molecules. The RGD (arginine‐glycine‐aspartate tripeptide motif)‐dependent integrin α8β1 has been shown to be involved in various cell functions in neuronal and mesenchymal‐derived cell types. Its role in epithelial cells remains unknown. Results. Integrin α8β1 was found to be expressed in the crypt cell population of the human intestine but was absent from differentiating and mature epithelial cells of the villus. The function of α8β1 in epithelial crypt cells was investigated at the cellular level using normal HIECs (human intestinal epithelial cells). Specific knockdown of α8 subunit expression using an shRNA (small‐hairpin RNA) approach showed that α8β1 plays important roles in RGD‐dependent cell adhesion, migration and proliferation via a RhoA/ROCK (Rho‐associated kinase)‐dependent mechanism as demonstrated by active RhoA quantification and pharmacological inhibition of ROCK. Moreover, loss of α8β1, through RhoA/ROCK, impairs FA (focal adhesion) complex integrity as demonstrated by faulty vinculin recruitment. Conclusions. Integrin α8β1 is expressed in epithelial cells. In intestinal crypt cells, α8β1 is closely involved in the regulation of adhesion, migration and cell proliferation via a predominant RhoA/ROCK‐dependent mechanism. These results suggest an important role for this integrin in intestinal crypt cell homoeostasis.