Serine-like proteolytic enzymes correlated with differential pathogenicity in patients with acute Acanthamoeba keratitis

P>Acute ocular infection due to free-living amoebae of the genus Acanthamoeba is characterized by severe pain, loss of corneal transparency and, eventually, blindness. Proteolytic enzymes secreted by trophozoites of virulent Acanthamoeba strains have an essential role in the mechanisms of pathogenesis, including adhesion, invasion and destruction of the corneal stroma. In this study, we analysed the relationship between the extracellular proteases secreted by clinical isolates of Acanthamoeba and the clinical manifestations and severity of disease that they caused. Clinical isolates were obtained from patients who showed typical symptoms of Acanthamoeba keratitis. Trophozoites were cultivated axenically, and extracellular proteins were collected from cell culture supernatants. Secreted enzymes were partially characterized by gelatin and collagen zymography. Acanthamoeba trophozoites secreted proteases with different molecular masses, proteolysis rates and substrate specificities, mostly serine-like proteases. Different enzymatic patterns of collagenases were observed, varying between single and multiple collagenolytic activities. Low molecular weight serine proteases were secreted by trophozoites associated with worse clinical manifestations. Consequently, proteolytic enzymes of some Acanthamoeba trophozoites could be related to the degree of their virulence and clinical manifestations of disease in the human cornea.

Cathepsin L-like genes of Trypanosoma vivax from Africa and South America - characterization, relationships and diagnostic implications

We characterized sequences from genes encoding cathepsin L-like (CatL-like) cysteine proteases from African and South American isolates of Trypanosoma vivax and T. vivax-like organisms, and evaluated their suitability as genetic markers for population structure analysis and diagnosis. Phylogenetic analysis of sequences corresponding to CatL-like catalytic domains revealed substantial polymorphism, and clades of sequences (TviCatL1-9) were separated by large genetic distances. TviCatL1-4 sequences were from cattle isolates from West Africa (Nigeria and Burkina Faso) and South America (Brazil and Venezuela), which belonged to the same T. vivax genotype. T. vivax-like genotypes from East Africa showed divergent sequences, including TviCatL5-7 for isolates from Mozambique and TviCatL8-9 for an isolate from Kenya. Phylogenetic analysis of CatL-like gene data supported the relationships among trypanosome species reflected in the phylogenies based on the analysis of small subunit (SSU) of ribosomal RNA gene sequence data. The discovery of different CatL-like sequences for each genotype, defined previously by ribosomal DNA data, indicate that these sequences provide useful targets for epidemiological and population genetic studies. Regions in CatL-like sequences shared by all T. vivax genotypes but not by other trypanosomes allowed the establishment of a specific and sensitive diagnostic PCR for epidemiological studies in South America and Africa. (C) 2008 Elsevier Ltd. All rights reserved.

Variants in the toll-like receptor signaling pathway and clinical outcomes of malaria

Background. Malaria is one of the most significant infectious diseases in the world and is responsible for a large proportion of infant deaths. Toll-like receptors (TLRs), key components of innate immunity, are central to countering infection. Variants in the TLR-signaling pathway are associated with susceptibility to infectious diseases. Methods. We genotyped single nucleotide polymorphisms ( SNPs) of the genes associated with the TLR-signaling pathway in patients with mild malaria and individuals with asymptomatic Plasmodium infections by means of polymerase chain reaction. Results. Genotype distributions for the TLR-1 I602S differed significantly between patients with mild malaria and persons with asymptomatic infection. The TLR-1 602S allele was associated with an odds ratio ( OR) of 2.2 ( P = .003; P(corrected) = .015) for malaria among patients with mild malaria due to any Plasmodium species and 2.1 ( P = .015; P(corrected) = .75) among patients with mild malaria due to Plasmodium falciparum only. The TLR-6 S249P SNP showed an excess of homozygotes for the TLR-6 249P allele in asymptomatic persons, compared with patients with mild malaria due to any Plasmodium species (OR 2.1; 95% confidence interval [CI], 1.1-4.2; P = .01; P(corrected) = .05), suggesting that the TLR-6 249S allele may be a risk factor for malaria ( OR, 2.0; 95% CI, 1.1-3.7; P = 0.01; P(corrected) = .05). The TLR-9-1486C allele showed a strong association with high parasitemia ( P < .001). Conclusions. Our findings indicate that the TLR-1 and TLR- 6 variants are significantly associated with mild malaria, whereas the TLR-9-1486C/T variants are associated with high parasitemia. These discoveries may bring additional understanding to the pathogenesis of malaria.

Phylogenetic analysis of Trypanosoma vivax supports the separation of South American/West African from East African isolates and a new T-vivax-like genotype infecting a nyala antelope from Mozambique

In this study, we addressed the phylogenetic and taxonomic relationships of Trypanosoma vivax and related trypanosomes nested in the subgenus Duttonella through combined morphological and phylogeographical analyses. We previously demonstrated that the clade T. vivax harbours a homogeneous clade comprising West African/South American isolates and the heterogeneous East African isolates. Herein we characterized a trypanosome isolated from a nyala antelope (Tragelaphus angasi) wild-caught in Mozambique (East Africa) and diagnosed as T. vivax-like based on biological, morphological and molecular data. Phylogenetic relationships, phylogeographical patterns and estimates of genetic divergence were based on SSU and ITS rDNA sequences of T. vivax from Brazil and Venezuela (South America), Nigeria (West Africa), and from T. vivax-like trypanosomes from Mozambique, Kenya and Tanzania (East Africa). Despite being well-supported within the T. vivax clade, the nyala trypanosome was highly divergent from all other T. vivax and T. vivax-like trypanosomes, even those from East Africa. Considering its host origin, morphological features, behaviour in experimentally infected goats, phylogenetic placement, and genetic divergence this isolate represents a new genotype of trypanosome closely phylogenetically related to T. vivax. This study corroborated the high complexity and the existence of distinct genotypes yet undescribed within the subgenus Duttonella.

Study of the mechanism of action of anoplin, a helical antimicrobial decapeptide with ion channel-like activity, and the role of the amidated C-terminus

Anoplin, an antimicrobial, helical decapeptide from wasp venom, looses its biological activities by mere deamidation of its C-terminus. Secondary structure determination, by circular dichroism spectroscopy in amphipathic environments, and lytic activity in zwitterionic and anionic vesicles showed quite similar results for the amidated and the carboxylated forms of the peptide. The deamidation of the C-terminus introduced a negative charge at an all-positive charged peptide, causing a loss of amphipathicity, as indicated by molecular dynamics simulations in TFE/water mixtures and this subtle modification in a peptide`s primary structure disturbed the interaction with bilayers and biological membranes. Although being poorly lytic, the amidated form, but not the carboxylated, presented ion channel-like activity on anionic bilayers with a well-defined conductance step; at approximately the same concentration it showed antimicrobial activity. The pores remain open at trans-negative potentials, preferentially conducting cations, and this situation is equivalent to the interaction of the peptide with bacterial membranes that also maintain a high negative potential inside. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells

Carraro-Lacroix LR, Malnic G, Girardi AC. Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells. Am J Physiol Renal Physiol 297: F1647-F1655, 2009. First published September 23, 2009; doi:10.1152/ajprenal.00082.2009.-The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na(+)/H(+) exchanger NHE3 in LLC-PK(1) cells. We found that NHE3-mediated Na(+)-dependent intracellular pH (pH(i)) recovery decreased similar to 50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pHi recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

The Thyroid Hormone Receptor (TR) beta-Selective Agonist GC-1 Inhibits Proliferation But Induces Differentiation and TR beta mRNA Expression in Mouse and Rat Osteoblast-Like Cells

Previous studies showed anabolic effects of GC-1, a triiodothyronine (T3) analogue that is selective for both binding and activation functions of thyroid hormone receptor (TR) beta 1 over TR alpha 1, on bone tissue in vivo. The aim of this study was to investigate the responsiveness of rat (ROS17/2.8) and mouse (MC3T3-E1) osteoblast-like cells to GC-1. As expected, T3 inhibited cellular proliferation and stimulated mRNA expression of osteocalcin or alkaline phosphatase in both cell lineages. Whereas equimolar doses of T3 and GC-1 equally affected these parameters in ROS17/2.8 cells, the effects of GC-1 were more modest compared to those of T3 in MC3T3-E1 cells. Interestingly, we showed that there is higher expression of TR alpha 1 than TR beta 1 mRNA in rat (similar to 20-90%) and mouse (similar to 90-98%) cell lineages and that this difference is even higher in mouse cells, which highlights the importance of TR alpha 1 to bone physiology and may partially explain the modest effects of GC-1 in comparison with T3 in MC3T3-E1 cells. Nevertheless, we showed that TR beta 1 mRNA expression increases (similar to 2.8- to 4.3-fold) as osteoblastic cells undergo maturation, suggesting a key role of TR beta 1 in mediating T3 effects in the bone forming cells, especially in mature osteoblasts. It is noteworthy that T3 and GC-1 induced TR beta 1 mRNA expression to a similar extent in both cell lineages (similar to 2- to 4-fold), indicating that both ligands may modulate the responsiveness of osteoblasts to T3. Taken together, these data show that TR beta selective T3 analogues have the potential to directly induce the differentiation and activity of osteoblasts.

A GRADIENT-LIKE NONAUTONOMOUS EVOLUTION PROCESS

In this paper we consider a dissipative damped wave equation with nonautonomous damping of the form u(tt) + beta(t)u(t) - Delta u + f(u) (1) in a bounded smooth domain Omega subset of R(n) with Dirichlet boundary conditions, where f is a dissipative smooth nonlinearity and the damping beta : R -> (0, infinity) is a suitable function. We prove, if (1) has finitely many equilibria, that all global bounded solutions of (1) are backwards and forwards asymptotic to equilibria. Thus, we give a class of examples of nonautonomous evolution processes for which the structure of the pullback attractors is well understood. That complements the results of [Carvalho & Langa, 2009] on characterization of attractors, where it was shown that a small nonautonomous perturbation of an autonomous gradient-like evolution process is also gradient-like. Note that the evolution process associated to (1) is not a small nonautonomous perturbation of any autonomous gradient-like evolution processes. Moreover, we are also able to prove that the pullback attractor for (1) is also a forwards attractor and that the rate of attraction is exponential.