Humoral immune response against soluble and fractionate antigens in experimental sporotrichosis

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii, which is widely distributed in nature, and presents a saprophytic mycelial form on plant debris and soil. The immunological mechanisms involved in the prevention and control of sporotrichosis are not yet fully understood. In this study, mice were studied after infection with Sporothrix schenckii. In the first week after infection, fungal loading increased and thence decreased drastically 14 days after infection. Analysis by immunoblotting showed that the sera of all mice tested had antibodies reacting only with a 70 kDa antigen, with predominance of IgG1 and IgG3. Taken together, our results show that antigens from S. schenckii induced a specific humoral response in infected mice.     

Taenia saginata: Production and characterization of monoclonal antibodies against Taenia saginata metacestode antigens

Cysticercosis is a major cause of economic loss in bovine production due to meat condemnation. Chemotherapy is being used in Brazilian cattle and a diagnostic test to improve the treatment program is desired. We produced monoclonal antibodies against crude (TAEB) and cyst fluid (TAEF) Taenia saginata metacestode antigens using immunized BALB/c mice. After cell fusion, 10 TAEB and nine TAEF hybrids were selected and cloned resulting in 18 IgG₁ and 32 IgM TAEB clones, and 9 IgG₁ and 9 IgM TAEF clones. Ascites was produced and Western blot testing was performed resulting in reactivity to protein fractions of low molecular weight (<18 kDa), 43, 55, 66 and 100 kDa. The indirect immunofluorescence test, with one monoclonal antibody against crude and one against cyst fluid antigens, recognized antigenic fractions of both the scolex and the bladder wall of metacestodes from naturally infected bovine.     

Modulation of the exocellular serine-thiol proteinase activity of Paracoccidioides brasiliensis by neutral polysaccharides

Our group characterized an exocellular serine-thiol proteinase activity in the yeast phase of Paracoccidioides brasiliensis (PbST), a dimorphic human pathogen. The fungal proteinase is able to cleave in vitro, at pH 7.4, proteins associated with the basal membrane, such as human laminin and fibronectin, type IV collagen and proteoglycans. In the present study, we investigated the influence of glycosaminoglycans (GAGs) and neutral polysaccharides upon the serine-thiol proteinase activity by means of kinetic analysis monitored with fluorescence resonance energy transfer (FRET) peptides using the substrate Abz-MKALTLQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp=ethylenediaminedinitrophenyl). Only neutral polysaccharides exhibited patterns of interaction with the proteinase, while sulfated GAGs had no effect. Incubation with neutral polysaccharides resulted in a powerful modulation of the enzyme activity, intensely changing the enzyme kinetic parameters of catalysis and affinity for the substrate. Commercial dextran at the highest concentration of 20 microM increased 6.8-fold the enzyme affinity for the substrate. In the presence of 8 microM of purified baker's yeast mannan, the apparent KM of the enzyme increased about 5.5-fold, reflecting a significant inhibition in binding to the peptide substrate. When an exocellular galactomannan (GalMan) complex isolated from P. brasiliensis was added to the reaction mixture at 400 nM, the apparent KM and VMAX decreased about threefold. Moreover, GalMan was able to protect the enzymatic activity at high temperatures, but it caused no effect on the optimum cleavage pH. Our results show a novel modulation mechanism in P. brasiliensis, where a fungal polysaccharide-rich component can stabilize a serine-thiol proteolytic activity, which is possibly involved in fungal dissemination.     

Role of NPR-C natriuretic receptor in nitric oxide system activation induced by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) exerts its hypotensive, natriuretic and diuretic effects, almost in part, through the activation of nitric oxide synthase (NOS). The aim was to investigate the natriuretic receptor type and the signaling cascade involved in NOS activation induced by ANP. Male Wistar rats were sacrificed and NOS activity was determined in kidney, aorta and heart with L-[U14C]-arginine, as substrate. ANP and cANP (4-23), a selective NPR-C ligand, increased NOS activity in all tissues. ANP induced a more marked activation in aorta and kidney than cANP (4-23), but no difference in atria NOS activation was observed. NOS activity induced by both peptides was blunted by nifedipine (L-type channel blocker) and calmidazolium (calmodulin antagonist) in heart and aorta. In kidney, nifedipine and calmidazolium abolished NOS activity stimulated by cANP (4-23) but only partially inhibited NOS activity elicited by ANP. Gi inhibition with pertussis toxin abolished NOS activity stimulated by ANP and cANP in atria but only partially inhibited the increased NOS activity induced by ANP and cANP in kidney, aorta and ventricle. Our results show that NPR-C receptor would mediate the activation of NOS by ANP in atria. In kidney, aorta and ventricle, NOS activation would also involve NPR-A and/or B. ANP would interact with NPR-C coupled via Gi to activation Ca2+ -dependent NOS.     

Arylfurans as potential trypanosoma cruzi trypanothione reductase inhibitors

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 microM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60% at 20 microg/ml (59 and 90 microM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 microg/ml (0.6-1.5 mM).     

Modeling human mitochondrial diseases in flies

Human mitochondrial diseases are associated with a wide range of clinical symptoms, and those that result from mutations in mitochondrial DNA affect at least 1 in 8500 individuals. The development of animal models that reproduce the variety of symptoms associated with this group of complex human disorders is a major focus of current research. Drosophila represents an attractive model, in large part because of its short life cycle, the availability of a number of powerful techniques to alter gene structure and regulation, and the presence of orthologs of many human disease genes. We describe here Drosophila models of mitochondrial DNA depletion, deafness, encephalopathy, Freidreich's ataxia, and diseases due to mitochondrial DNA mutations. We also describe several genetic approaches for gene manipulation in flies, including the recently developed method of targeted mutagenesis by recombinational knock-in.     

Syndecan-4 regulates non-canonical Wnt signalling and is essential for convergent and extension movements in Xenopus embryos

Early shaping of Xenopus laevis embryos occurs through convergent and extension movements, a process that is driven by intercalation of polarized dorsal mesodermal cells and regulated by non-canonical Wnt signalling. Here, we have identified Xenopus syndecan-4 (xSyn4), a cell-surface transmembrane heparan sulphate proteoglycan. At the gastrula stage, xSyn4 is expressed in the involuting dorsal mesoderm and the anterior neuroectoderm. Later, it is found in the pronephros, branchial arches, brain and tailbud. Both gain- and loss-of-function of xSyn4 impaired convergent extension movements in Xenopus embryos and in activin-treated ectodermal explants. xSyn4 interacts functionally and biochemically with the Wnt receptor Frizzled7 (xFz7) and its signal transducer Dishevelled (xDsh). Furthermore, xSyn4 is necessary and sufficient for translocation of xDsh to the plasma membrane - a landmark in the activation of non-canonical Wnt signalling. Our results suggest that the ability of xSyn4 to translocate xDsh is regulated by fibronectin, a component of the extracellular matrix required for proper convergent extension movements. We propose a model where xSyn4 and fibronectin cooperate with xFz7 and Wnt in the specific activation of the non-canonical Wnt pathway.     

A microsatellite-based consensus linkage map for species of Eucalyptus and a novel set of 230 microsatellite markers for the genus

Background  

Eucalypts are the most widely planted hardwood trees in the world occupying globally more than 18 million hectares as an important source of carbon neutral renewable energy and raw material for pulp, paper and solid wood. Quantitative Trait Loci (QTLs) in Eucalyptus have been localized on pedigree-specific RAPD or AFLP maps seriously limiting the value of such QTL mapping efforts for molecular breeding. The availability of a genus-wide genetic map with transferable microsatellite markers has become a must for the effective advancement of genomic undertakings. This report describes the development of a novel set of 230 EMBRA microsatellites, the construction of the first comprehensive microsatellite-based consensus linkage map for Eucalyptus and the consolidation of existing linkage information for other microsatellites and candidate genes mapped in other species of the genus.     

Carbohydrate- and lipid-enriched meals acutely disrupt glycemic homeostasis by inducing transient insulin resistance in rats

Chronic intake of high-carbohydrate or high-lipid diets is a well-known insulin resistance inducer. This study investigates the immediate effect (1-6 h) of a carbohydrate- or lipid-enriched meal on insulin sensitivity. Fasted rats were refed with standard, carbohydrate-enriched (C), or lipid-enriched (L) meal. Plasma insulin, glucose, and non-esterified fatty acids (NEFA) were measured at 1, 2, 4, and 6 h of refeeding. The glucose-insulin index showed that either carbohydrates or lipids decreased insulin sensitivity at 2 h of refeeding. At this time point, insulin tolerance tests (ITTs) and glucose tolerance tests (GTTs) detected insulin resistance in C rats, while GTT confirmed it in L rats. Reduced glycogen and phosphorylated AKT and GSK3 content revealed hepatic insulin resistance in C rats. Reduced glucose uptake in skeletal muscle subjected to the fatty acid concentration that mimics the high NEFA level of L rats suggests insulin resistance in these animals is mainly in muscle. In conclusion, carbohydrate- or lipid-enriched meals acutely disrupt glycemic homeostasis, inducing a transient insulin resistance, which seems to involve liver and skeletal muscle, respectively. Thus, the insulin resistance observed when those types of diets are chronically consumed may be an evolution of repeated episodes of this transient insulin resistance.