Purification of a lectin from the marine red alga Gracilaria ornata and its effect on the development of the cowpea weevil Callosobruchus maculatus (Coleoptera: Bruchidae)

A lectin from the marine red alga Gracilaria ornata (Gracilariaceae, Rodophyta) was purified and characterized. The purification procedure consisted of extracting soluble proteins in 0.025 M Tris-HCl buffer, pH 7.5, followed by ammonium sulfate precipitation (70% saturation), ion exchange chromatography on DEAE-cellulose and affinity chromatography on mucin-Sepharose 4B. The purified G. ornata lectin (GOL) showed a single protein band with an apparent molecular mass of 17 kDa when submitted to SDS-polyacrylamide gel electrophoresis under reducing conditions. The native molecular mass of GOL determined by gel filtration on a Sephadex G-100 column was 17.4 kDa and its carbohydrate content was estimated to be 2.9%. Therefore, GOL is a monomeric glycoprotein. The purified lectin agglutinated trypsin-treated erythrocytes from rabbit and chicken but not from human. Its activity was not inhibited by any of the mono- and disaccharides tested but by the complex glycoproteins porcine stomach mucin, lactotransferrin, asialofetuin and bovine and porcine thyroglobulins. Isoelectric focusing showed that GOL is an acidic protein with a pI of 5.4 with analysis of its amino acid composition revealing high contents of Asx, Glx, Ser, Glu, Ala and Cys. When incorporated in artificial seeds, GOL significantly affected the development of Callosobruchus maculatus larvae, indicating the possibility of using this lectin in a biotechnological strategy for insect management of stored cowpea seeds.     

De novo methylation of nucleosomal DNA by the mammalian Dnmt1 and Dnmt3A DNA methyltransferases

In the cell, DNA is wrapped on histone octamers, which reduces its accessibility for DNA interacting enzymes. We investigated de novo methylation of nucleosomal DNA in vitro and show that the Dnmt3a and Dnmt1 DNA methyltransferases efficiently methylate nucleosomal DNA without dissociation of the histone octamer from the DNA. In contrast, the prokaryotic SssI DNA methyltransferase and the catalytic domain of Dnmt3a are strongly inhibited by nucleosomes. We also found that full-length Dnmt1 and Dnmt3a bind to nucleosomes much stronger than their isolated catalytic domains, demonstrating that the N-terminal parts of the MTases are required for the interaction with nucleosomes. Variations of the DNA sequence or the histone tails did not significantly influence the methylation activity of Dnmt3a. The observation that mammalian methyltransferases directly modify nucleosomal DNA provides an insight into the mechanisms by which histone tail and DNA methylation patterns can influence each other because the DNA methylation pattern can be established while histones remain associated to the DNA.     

The modelling of mononuclear phagocyte-connective tissue adhesion in vitro: application to disclose a specific inhibitory effect of Leishmania infection

In this work, we have developed an adhesion assay to study interactions between mononuclear phagocytes and connective tissue in vitro and show its potential use to study diseases caused by intracellular microorganisms. The assay reproduces most of the characteristics of macrophage adhesion to connective tissue in vivo, such as: preferential adhesion to inflamed connective tissue, divalent cation and integrin dependence, and up-regulation upon cell activation. The phagocyte adhesion to connective tissue was inhibited by infection with Leishmania (58+/-22%, p < 0.05) and was not affected by infection with Mycobacterium or by endocytosis of latex beads. Manganese partially reverted the loss in adherence produced by Leishmania infection, indicating that the mechanisms regulating the function of integrins are affected by cell infection with Leishmania. This assay might be a useful tool for the study of the mechanisms by which mononuclear phagocytes play a role in the immune-inflammatory response and in the development of lesions.     

Chiral evaluation of fluvastatin in human plasma by high-performance liquid chromatography electrospray mass spectrometry

The report describes for the first time the enantioselective analysis of fluvastatin in plasma using LC-MS-MS. The enantiomers of fluvastatin (FV) were extracted from plasma with diisopropyl ether at pH 5.0. The enantiomers were separated on a ChiralCel OD-R column with a mobile phase consisting of a mixture of acetonitrile, methanol and water (24:36:40) containing 0.1% formic acid. The protonated ions and their respective product ions were monitored in two functions, 410.6>348.2 for FV enantiomers and 307.1>161.6 for the internal standard (warfarin). Recoveries were higher than 90% and the quantitation limit was 1.5 ng mL(-1) plasma for both enantiomers. The coefficients of variation and the relative errors obtained for the validation of the intra- and interassay precision and accuracy were less than 10%. The method was applied to the investigation of the enantioselective pharmacokinetics of FV administered in a single dose of 40 mg (Lescol, Novartis, S?o Paulo, SP, Brazil) to a patient with primary hypertension and hypercholesterolemia and genotyped as CYP2C9*1/*1. The data showed higher plasma concentrations of the (-)-3S,5R-fluvastatin enantiomer, with an AUC (-)/(+) of 1.84. Oral clearance values (CL/F) were 29.27 and 49.58 L/h, respectively, for the (-)-3S,5R- and (+)-3R,5S-fluvastatin enantiomers.     

Nitrate uptake and utilization is modulated by exogenous γ-aminobutyric acid in Arabidopsis thaliana seedlings

Exogenously applied GABA modulates root growth by inhibition of root elongation when seedlings were grown in vitro on full-strength Murashige and Skoog (MS) salts, but root elongation was stimulated when seedlings were grown on 1/8 strength MS salts. When the concentration of single ions in MS salts was individually varied, the control of growth between inhibition and stimulation was found to be related to the level of nitrate (NO₃^?) in the growth medium. At NO₃^? concentrations below 40 mM (full-strength MS salts level), root growth was stimulated by the addition of GABA to the growth medium; whereas at concentrations above 40 mM NO₃^?, the addition of GABA to the growth medium inhibited root elongation. GABA promoted NO₃^? uptake at low NO₃^?, while GABA inhibited NO₃^? uptake at high NO₃^?. Activities of several enzymes involved in nitrogen and carbon metabolism including nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (NADH-GOGAT), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and phosphoenol pyruvate carboxylase (PEPCase) were regulated by GABA in the growth medium. Supplementing 1/8 strength MS medium with 50 mM GABA enhanced the activities of all of the above enzymes except ICDH activities in root tissues. However, at full-strength MS, GABA showed no inhibitory effect on the activities of these enzymes, except on GS in both root and shoot tissues, and PEPCase activity in shoot tissues. Exogenous GABA increased the amount of NR protein rather than its activation status in the tissues. This study shows that GABA affects the growth of Arabidopsis, possibly by acting as a signaling molecule, modulating the activity of enzymes involved in primary nitrogen metabolism and nitrate uptake.     

Evaluating Tropical Biodiversity: Do We Need a More Refined Approach?

The exceptional biodiversity of tropical forests inspired the earliest ecologists such as H. W. Bates. Today we still strive to quantify and understand this diversity. Drawing on our own experience of Mamirauá reserve in Amazonas, which is located in an area that Bates explored, we argue that the emphasis of research in tropical ecosystems should shift away from species richness as an end in itself, and focus instead on other fundamental, but more tractable, questions that will increase our ecological understanding of these systems, support conservation management, and appeal to policy makers and society in general.     

Leptin effect on RANKL and OPG expression in MC3T3-E1 osteoblasts

Recent studies have suggested that leptin hormone may play a pivotal role on bone remodeling through a direct effect by modulating positively the OPG/RANKL balance. Here, we investigate the effect of leptin hormone on RANKL and OPG expression in MC3T3-E1 osteoblasts using RT-PCR and ELISA measurements. We have at first identified the expression of Ob-Rb and Ob-Ra leptin receptor isoforms in MC3T3-E1 and observed that these cells respond to mrleptin treatments. We then investigated the effect of mrleptin on RANKL and OPG expression. We show that mrleptin dose-dependently regulated the expression of RANKL mRNA with complete inhibition observed at concentrations higher than 12 ng/ml. This effect was confirmed with sRANKL protein measurements. However, the exposure of MC3T3-E1 to mrleptin had no effect on OPG mRNA. Taken together, these results suggest that leptin modulates positively OPG/RANKL balance by inhibiting the expression of RANKL gene.     

Genetic transformation of Rangpur lime (Citrus limonia osbeck) with thebO (bacterio-opsin) genen and its initial evaluation forPhytophthora nicotianae resistance

Transgenic plants expressing the bacterio-opsin (bO) gene can spontaneously activate programmed cell death (ped) and may enhance broad-spectrum pathogen resistance by activating an intrinsic defense pathway in plant species such as tobacco and potato. In this work, we produced transgenic Rangpur lime plants with thebO gene, viaAgrobacterium tumefaciens-mediated transformation, and evaluated these plants forPhytophthora nicotianae resistance. Two transgenic lines were successfully regenerated and transformation was confirmed by GUS activity assay, PCR analysis, Southern, Northern and Western blot analyses, in addition to detecting the expressed bO protein by an immunological approach. Evaluation forPhytophthora nicotianae resistance was carried out by plant inoculations with the pathogen and quantification of the affected area. One of the two transgenic lines showed greater tolerance to the fungal pathogen as compared to the control, with significantly smaller stem lesions after pathogen challenge. This increase in pathogen tolerance is correlated with a significantly higher level of transgene expression in this line when compared with the other transgenic line. This is the first report of the introduction of a potentially important gene into Rangpur lime to provide novel pathogen tolerance.     

Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown Clades

Inference of evolutionary relationships between nematodes is severely hampered by their conserved morphology, the high frequency of homoplasy, and the scarcity of phylum-wide molecular data. To study the origin of nematode radiation and to unravel the phylogenetic relationships between distantly related species, 339 nearly full-length small-subunit rDNA sequences were analyzed from a diverse range of nematodes. Bayesian inference revealed a backbone comprising 12 consecutive dichotomies that subdivided the phylum Nematoda into 12 clades. The most basal clade is dominated by the subclass Enoplia, and members of the order Triplonchida occupy positions most close to the common ancestor of the nematodes. Crown Clades 8-12, a group formerly indicated as "Secernentea" that includes Caenorhabditis elegans and virtually all major plant and animal parasites, show significantly higher nucleotide substitution rates than the more basal Clades 1-7. Accelerated substitution rates are associated with parasitic lifestyles (Clades 8 and 12) or short generation times (Clades 9-11). The relatively high substitution rates in the distal clades resulted in numerous autapomorphies that allow in most cases DNA barcode-based species identification. Teratocephalus, a genus comprising terrestrial bacterivores, was shown to be most close to the starting point of Secernentean radiation. Notably, fungal feeding nematodes were exclusively found basal to or as sister taxon next to the 3 groups of plant parasitic nematodes, namely, Trichodoridae, Longidoridae, and Tylenchomorpha. The exclusive common presence of fungivorous and plant parasitic nematodes supports a long-standing hypothesis that states that plant parasitic nematodes arose from fungivorous ancestors.