Evaluation of jackknife and bootstrap for defining confidence intervals for pairwise agreement measures

Several research fields frequently deal with the analysis of diverse classification results of the same entities. This should imply an objective detection of overlaps and divergences between the formed clusters. The congruence between classifications can be quantified by clustering agreement measures, including pairwise agreement measures. Several measures have been proposed and the importance of obtaining confidence intervals for the point estimate in the comparison of these measures has been highlighted. A broad range of methods can be used for the estimation of confidence intervals. However, evidence is lacking about what are the appropriate methods for the calculation of confidence intervals for most clustering agreement measures. Here we evaluate the resampling techniques of bootstrap and jackknife for the calculation of the confidence intervals for clustering agreement measures. Contrary to what has been shown for some statistics, simulations showed that the jackknife performs better than the bootstrap at accurately estimating confidence intervals for pairwise agreement measures, especially when the agreement between partitions is low. The coverage of the jackknife confidence interval is robust to changes in cluster number and cluster size distribution.     

Thermodynamic properties and characterization of proteoliposomes rich in microdomains carrying alkaline phosphatase

Tissue-nonspecific alkaline phosphatase (TNAP) is associated to the plasma membrane via a GPI-anchor and plays a key role in the biomineralization process. In plasma membranes, most GPI-anchored proteins are associated with "lipid rafts", ordered microdomains enriched in sphingolipids, glycosphingolipids and cholesterol. In order to better understand the role of lipids present in rafts and their interactions with GPI-anchored proteins, the insertion of TNAP into different lipid raft models was studied using dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), sphingomyelin (SM) and ganglioside (GM1). Thus, the membrane models studied were binary systems (9:1 molar ratio) containing DPPC:Chol, DPPC:SM and DPPC:GM1, ternary systems (8:1:1 molar ratio) containing DPPC:Chol:SM, DPPC:Chol:GM1 and DPPC:SM:GM1 and finally, a quaternary system (7:1:1:1 molar ratio) containing DPPC:Chol:SM:GM1. Calorimetry analysis of the liposomes and proteoliposomes indicate that lateral phase segregation could be noted only in the presence of cholesterol, with the formation of cholesterol-rich microdomains centered above Tc=41.5°C. The presence of GM1 and SM into DPPC-liposomes influenced mainly ΔH and Δt(1/2) values. The gradual increase in the complexity of the systems decreased the activity of the enzyme incorporated. The presence of the enzyme also fluidifies the systems, as seen by the intense reduction in ?H values, but do not alter Tc values significantly. Therefore, the study of different microdomains and its biophysical characterization may contribute to the knowledge of the interactions between the lipids present in MVs and its interactions with TNAP.     

Evolved streptavidin mutants reveal key role of loop residue in high-affinity binding

We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a ～10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions.     

Aquaporin inhibition changes protein phosphorylation pattern following sperm motility activation in fish

Our previous studies demonstrated that osmolality is the key signal in sperm motility activation in Sparus aurata spermatozoa. In particular, we have proposed that the hyper-osmotic shock triggers water efflux from spermatozoa via aquaporins. This water efflux determines the cell volume reduction and, in turn, the rise in the intracellular concentration of ions. This increase could lead to the activation of adenylyl cyclase and of the cAMP-signaling pathway, causing the phosphorylation of sperm proteins and then the initiation of sperm motility. This study confirms the important role of sea bream AQPs (Aqp1a and Aqp10b) in the beginning of sperm motility. In fact, when these proteins are inhibited by HgCl₂, the phosphorylation of some proteins (174 kDa protein of head; 147, 97 and 33 kDa proteins of flagella), following the hyper-osmotic shock, was inhibited (totally or partially). However, our results also suggest that more than one transduction pathways could be activated when sea bream spermatozoa were ejaculated in seawater, since numerous proteins showed an HgCl₂(AQPs)-independent phosphorylation state after motility activation. The role played by each different signal transduction pathways need to be clarified.     

The Hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.     

Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H₂O₂ affair?

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.     

Floristic composition and similarity of 15 hectares in Central Amazon, Brazil

The Amazon region is one of the most diverse areas in the world. Research on high tropical forest diversity brings up relevant contributions to understand the mechanisms that result and support such diversity. In the present study we describe the species composition and diversity of 15 one-ha plots in the Amazonian terra firme dense forest in Brazil, and compare the floristic similarity of these plots with other nine one-ha plots. The 15 plots studied were randomly selected from permanent plots at the Embrapa Experimental site, Amazonas State in 2005. The diversity was analysed by using species richness and Shannon's index, and by applying the Sorensen's index for similarity and unweighted pair-group average (UPGMA) as clustering method. Mantel test was performed to study whether the differences in species composition between sites could be explained by the geographic distance between them. Overall, we identified 8 771 individuals, 264 species and 51 plant families. Most of the species were concentrated in few families and few had large number of individuals. Families presenting the highest species richness were Fabaceae (Faboideae: 22spp., Mimosoideae: 22spp.), Sapotaceae: 22spp., Lecythidaceae: 15 and Lauraceae: 13. Burseraceae had the largest number of individuals with 11.8% of the total. The ten most abundant species were: Protium hebetatum (1 037 individuals), Eschweilera coriacea (471), Licania oblongifolia (310), Pouteria minima (293), Ocotea cernua (258), Scleronema micranthum (197), Eschweilera collina (176), Licania apelata (172), Naucleopsis caloneura (170) and Psidium araca (152), which represented 36.5% of all individuals. Approximately 49% of species had up to ten individuals and 13% appeared only once in all sampled plots, showing a large occurrence of rare species. Our study area is on a forest presenting a high tree species diversity with Shannon's diversity index of 4.49. The dendrogram showed two groups of plots with low similarity between them (less than 0.25), and the closer the plots were one to another, more similar in species composition (Mantel R = 0.3627, p < 0.01). The 15 plots in our study area share more than 50% of their species composition and represent the group of plots that have the shortest distance between each other. Overall, our results highlight the high local and regional heterogeneity of environments in terra firme forests, and the high occurrence of rare species, which should be considered in management and conservation programs in the Amazon rainforest, in order to maintain its structure on the long run.     

Nongenomic signaling pathways triggered by thyroid hormones and their metabolite 3‐iodothyronamine on the cardiovascular system

Thyroid hormones play a wide range of important physiological activities in almost all organism. As changes in these hormones levels—observed in hypothyroidism and hyperthyroidism—promote serious derangements of the cardiovascular system, it is important to know their mechanisms of action. Although the classic genomic actions which are dependent on interaction with nuclear receptors to modulate cardiac myocytes genes expression, there is growing evidence about T₃ and T₄‐triggered nongenomic pathways, resulted from their binding to plasma membrane, cytoplasm, or mitocondrial receptors that leads to a rapidly regulation of cardiac functions. Interestingly both actions converge to amplify thyroid hormone effects on cardiovascular system. T₃ and T₄ nongenomic actions modify inotropic and chronotropic effects, cardiac action potential duration, cardiac growth, and myocyte shape by protein translation through protein kinases‐dependent signaling cascades, which include PKA, PKC, PI3K, and MAPK, and changes on ion channels and pumps activity. In respect to the decreased systemic vascular resistance seen in hyperthyroidism, T₃ appears to activate NOS or ATP‐sensitive K⁺ channels. In addition, a novel biologically active T₄‐derived metabolite has been described, 3‐iodothyronamine, T₁AM, which also acts through membrane receptors to mediate nongenomic cardiac effects. This metabolite influences the physiological manifestations of thyroid hormone actions by inducing opposite effects from those stimulated by T₃ and T₄, such as negative inotropic and chronotropic effects. Therefore, beyond genomic and nongenomic effects of thyroid hormones, it is crucial for there to be an equilibrium between T₃ or T₄ and T₁AM levels for maintaining cardiac homeostasis. J. Cell. Physiol. 226: 21–28, 2010. © 2010 Wiley‐Liss, Inc.     

Cloning,expression, molecular modelling and docking analysis of glutathione transferase from Saccharum officinarum

Sugarcane yield and quality are affected by a number of biotic and abiotic stresses. In response to such stresses, plants may increase the activities of some enzymes such as glutathione transferase (GST), which are involved in the detoxification of xenobiotics. Thus, a sugarcane GST was modelled and molecular docked using the program LIGIN to investigate the contributions of the active site residues towards the binding of reduced glutathione (GSH) and 1‐chloro‐2,4‐dinitrobenzene (CDNB). As a result, W13 and I119 were identified as key residues for the specificity of sugarcane GSTF1 (SoGSTF1) towards CDNB. To obtain a better understanding of the catalytic specificity of sugarcane GST (SoGSTF1), two mutants were designed, W13L and I119F. Tertiary structure models and the same docking procedure were performed to explain the interactions between sugarcane GSTs with GSH and CDNB. An electron‐sharing network for GSH interaction was also proposed. The SoGSTF1 and the mutated gene constructions were cloned and expressed in Escherichia coli and the expressed protein purified. Kinetic analyses revealed different K_m values not only for CDNB, but also for GSH. The K_m values were 0.2, 1.3 and 0.3 mM for GSH, and 0.9, 1.2 and 0.5 mM for CDNB, for the wild type, W13L mutant and I119F mutant, respectively. The V_max values were 297.6, 224.5 and 171.8 µmol min^?1 mg^?1 protein for GSH, and 372.3, 170.6 and 160.4 µmol min^?1 mg^?1 protein for CDNB.     

Structural and biological characterization of Nattectin, a new C-type lectin from the venomous fish Thalassophryne nattereri

Lectins are glycan-binding receptors that recognize glycan epitopes on foreign pathogens and in the host systems. They can be involved in functions that include innate immunity, development, immune regulation and homeostasis. Several lectins have been purified and characterized from fish species. In this work, using cation-exchange chromatography, a galactose-specific lectin belonging to the family of C-type lectins was isolated from the venom of the Brazilian venomous fish Thalassophryne nattereri. Nattectin is a basic, non-glycosilated, 15 kDa monomeric protein. It exhibits hemagglutination activity that is independent of Ca²⁺. We also demonstrated a lectin activity for Nattectin in the innate immune system, especially in neutrophil mobilization in mice, indicating that marine organisms are source of immunomodulator agents.