Identification of enolase as a laminin-binding protein on the surface of Staphylococcus aureus

We have previously demonstrated that Staphylococcus aureus, a highly invasive bacteria, presents a 52-kDa surface protein that mediates its binding to laminin. In order to better characterize this receptor, we excised this putative laminin receptor from two-dimensional (2-D) PAGE and used it as antigen for raising a mouse hyperimmune serum which was for screening an S. aureus expression library. A single clone of 0.3 kb was obtained, and its sequence revealed 100% homology with S. aureus alpha-enolase. Moreover, amino acid sequencing of the 52-kDa protein eluted from the 2-D gel indicated its molecular homology with alpha-enolase, an enzyme that presents a high evolutionary conservation among species. In parallel, monoclonal antibodies raised against the S. aureus 52-kDa band also recognized yeast alpha-enolase in western blot analysis. These monoclonal antibodies were also able to promote capture of iodine-labeled bacteria when adsorbed to a solid phase, and this capture was inhibited by the addition of excess rabbit muscle alpha-enolase. Finally, the cell surface localization of S. aureus alpha-enolase was further confirmed by flow cytometry. Hence, alpha-enolase might play a critical role in the pathogenesis of S. aureus by allowing its adherence to laminin-containing extracellular matrix.     

Probing the location of the substrate binding site of ascorbate oxidase near type 1 copper: an investigation through spectroscopic, inhibition and docking studies

The present investigation addresses the problem of the binding mode of phenolic inhibitors and the substrate ascorbate to the active site of ascorbate oxidase. The results from both types of compounds indicate that the binding site is located in a pocket near the type 1 copper center. This information is of general interest for blue multicopper oxidases. Docking calculations performed on the ascorbate oxidase-ascorbate complex show that binding of the substrate occurs in a pocket near type 1 Cu, and is stabilized by at least five hydrogen bonding interactions with protein residues, one of which involves the His512 Cu ligand. Similar docking studies show that the isomeric fluorophenols, which act as competitive inhibitors toward ascorbate, bind to the enzyme in a manner similar to ascorbate. The docking calculations are supported by 19F NMR relaxation measurements performed on fluorophenols in the presence of the enzyme, which show that the bound inhibitors undergo enhanced relaxation by the paramagnetic effect of a nearby Cu center. Unambiguous support to the location of the inhibitor close to type 1 Cu was obtained by comparative relaxation measurements of the fluorophenols in the presence of the ascorbate oxidase derivative where a Zn atom selectively replaces the paramagnetic type 2 Cu. The latter experiments show that contribution to relaxation of the bound inhibitors by the type 2 Cu site is negligible.     

Role of peroxynitrite in macrophage microbicidal mechanisms in vivo revealed by protein nitration and hydroxylation

The cytotoxins produced by phagocytic cells lacking peroxidases such as macrophages remain elusive. To elucidate macrophage microbicidal mechanisms in vivo, we compared the lesion tissue responses of resistant (C57Bl/6) and susceptible (BALB/c) mice to Leishmania amazonensis infection. This comparison demonstrated that parasite control relied on lesion macrophage activation with inducible nitric oxide synthase expression (iNOS), nitric oxide synthesis, and extensive nitration of parasites inside macrophage phagolysosomes at an early infection stage. Nitration and iNOS expression were monitored by confocal microscopy; nitric oxide synthesis was monitored by EPR. The main macrophage nitrating agent was shown to be peroxynitrite derived because parasite nitration occurred in the virtual absence of polymorphonuclear cells (monitored as peroxidase activity) and was accompanied by protein hydroxylation (monitored as 3-hydroxytyrosine levels). In vitro studies confirmed that peroxynitrite is cytotoxic to parasites whereas nitric oxide is cytostatic. The results indicate that peroxynitrite is likely to be produced close to the parasites and most of it reacts with carbon dioxide to produce carbonate radical anion and nitrogen dioxide whose concerted action leads to parasite nitration. In parallel, some peroxynitrite decomposition to the hydroxyl radical should occur due to the detection of hydroxylated proteins in the healing tissues. Consequently, peroxynitrite and derived radicals are likely to be important macrophage-derived cytotoxins.     

Relative growth and morphological sexual maturity of the freshwater crab Trichodactylus fluviatilis Latreille 1828 (Decapoda,Trichodactylidae) from west central São Paulo State,Brazil

An analysis of relative growth was performed to determine the size at morphological sexual maturity of Trichodactylus fluviatilis Latreille 1828. Samples were collected by trapping at night from January 2010 through June 2011 from the Barreiro River (22°34′42″S; 49°11′92″W), west central São Paulo State, Brazil. The following measurements were obtained (mm): carapace width (CW) (independent variable), cheliped propodus length (PL), cheliped propodus height (PH), cheliped propodus width (PW), first pleopod length, and abdomen width (AW). The males showed positive allometry in the juvenile and adult stages for the following relationships: PL vs. CW, PH vs. CW, and PW vs. CW. In the females, a positive allometry was observed for AW vs. CW in the juvenile stage, and a negative allometry was observed for this relationship in the adult stage. The differential growth of the chelipeds in males could be related to territorial conflict and courtship, whereas the changes in the growth of the female abdomen most likely favor higher reproductive success.     

Alternative Prey and Abundance Covariance Switches an Intraguild Predator’s Functional Response

Positive or negative prey abundance covariances play an important role in determining prey preference of predators. The goal here was to understand how variations in abundance of two blowfly prey species, a native and a non-native species, influence the switching behavior and functional response of Chrysomya albiceps, an intraguild predatory blowfly, under laboratory conditions. The results suggest C. albiceps prefers to consume a native prey species rather than a non-native prey species. However, when prey densities covariate negatively, both species were consumed at the same rate, changing predator’s functional response from type II to type III. The conditions that trigger the switching behavior in blowfly communities are discussed in detail in this study.     

Influence of aeration–homogenization system in stirred tank bioreactors,dissolved oxygen concentration and pH control mode on BHK-21 cell growth and metabolism

This work focused on determining the effect of dissolved oxygen concentration (DO) on growth and metabolism of BHK-21 cell line (host cell for recombinant proteins manufacturing and viral vaccines) cultured in two stirred tank bioreactors with different aeration-homogenization systems, as well as pH control mode. BHK-21 cell line adapted to single-cell suspension was cultured in Celligen without aeration cage (rotating gas-sparger) and Bioflo 110, at 10, 30 and 50 % air saturation (impeller for gas dispersion from sparger-ring). The pH was controlled at 7.2 as far as it was possible with gas mixtures. In other runs, at 30 and 50 % (DO) in Bioflo 110, the cells grew at pH controlled with CO₂ and NaHCO₃ solution. Glucose, lactate, glutamine, and ammonium were quantified by enzymatic methods. Cell concentration, size and specific oxygen consumption were also determined. When NaHCO₃ solution was not used, the optimal DOs were 10 and 50 % air saturation for Celligen and Bioflo 110, respectively. In this condition maximum cell concentrations were higher than 4 × 10⁶ cell/mL. An increase in maximum cell concentration of 36 % was observed in batch carried out at 30 % air saturation in a classical stirred tank bioreactor (Bioflo 110) with base solution addition. The optimal parameters defined in this work allow for bioprocess developing of viral vaccines, transient protein expression and viral vector for gene therapy based on BHK-21 cell line in two stirred tank bioreactors with different agitation–aeration systems.     

MODULAR: software for the autonomous computation of modularity in large network sets

Many ecological systems can be represented as networks of interactions. A key feature in these networks is their organization into modules, which are subsets of tightly connected elements. We introduce MODULAR to perform rapid and autonomous calculation of modularity in network sets. MODULAR reads a set of files representing unipartite or bipartite networks, and identifies modules using two different modularity metrics widely used in the ecological networks literature. To estimate modularity, the software offers five optimization methods to the user. The software also includes two null models commonly used in studies of ecological networks to verify how the degree of modularity differs from two distinct theoretical benchmarks.     

Morphology of the unusual polyad in Amazonian Parkia legume trees

Key message

An unusual polyad occurs in three Parkia species, named cavitate polyad. It has an internal central space full of lipoprotein substances, contacting all pollen grains, probably aiding pollen germination.

Abstract

This study details the unusual morphology of polyads of three species of Parkia (P. multijuga Benth., P. ulei (Harms) Kuhlm., and P. pendula (Willd.) Benth. ex Walp.) and suggests functions for polyad adaptive traits that are linked to the reproductive success of the species. Polyads within the anthers of the three Parkia species were analysed by surface (scanning electron microscopy) and anatomical (light microscopy) studies. Ultrastructure and development studies were carried out for P. pendula polyads. Polyads are globose and cavitated, i.e., exhibit an internal cavity that varies in size, being more conspicuous in P. ulei and P. pendula. Other differences among species are related to the polyad size, exine ornamentation and the type of substances stored in the pollen grain. The polyad internal cavity is filled with an exudate that may be related to the pollen germination through the internal pores and/or translocation of substances from the anther loculus to the inside or vice versa. This inference is supported by the following observations: the spaces between the pollen grains in a polyad are also filled with the exudate, and the exudate inside the polyad is similar to the anther locular fluid. The morphology and substances stored within the pollen grains of Parkia polyads seem to be more related to polyad functionality and physiology than to the selective pressures exerted by different pollinators on the group.     

Taurine supplementation increases KATP channel protein content,improving Ca2+ handling and insulin secretion in islets from malnourished mice fed on a high-fat diet

Pancreatic β-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and K_ATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5 % Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced K_ATP inhibition and Cav activity. RH islets secreted less insulin at high K⁺ concentration and showed enhanced K_ATP activity. Tau supplementation normalized K⁺-induced secretion and enhanced glucose-induced Ca²⁺ influx in RHT islets. R islets presented lower Ca²⁺ influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the K_ATP. Tau increased the protein content of the α1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the K_ATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the K_ATP channels and enhancing Synt-1 islet content.