Effect of pre-exposure of human erythrocytes to oxidants on the haemolytic activity of Sticholysin II. A comparison between peroxynitrite and hypochlorous acid

Stichodactyla heliantus II (St II) is a haemolytic toxin whose activity depends of the characteristics of red blood cells (RBC). Among the factors that may tune the response of the RBC to the toxin activity stand the oxidative status of the cell. This study investigates how pre-oxidation of RBC modifies St II activity employing two oxidants, peroxynitrite and hypochlorous acid. Results show that peroxynitrite-treated RBC are more resistant to St II activity. On the other hand, hypochlorous acid-treated RBC become more susceptible to St II. This contrasting behaviour of both oxidants is related to the modifications elicited in RBC by both oxidant agents. Peroxynitrite does not modify RBC osmotic fragility but reduces anion transport through band 3 protein. This effect, together with an increase in K+ efflux, can explain the increased resistance to the toxin activity. On the other hand, results obtained with hypochlorous acid can be explained in terms of a disruption of the membrane organization without the compensating effect of a reduction in band 3-mediated anion transport. The present results, obtained employing the effect of a model haemolytic toxin on RBC, emphasize the specificity of the RBC response to different endogenous oxidative agents.     

Obestatin as a regulator of adipocyte metabolism and adipogenesis

The role of obestatin, a 23-amino-acid peptide encoded by the ghrelin gene, on the control of the metabolism of pre-adipocyte and adipocytes as well as on adipogenesis was determined. For in vitro assays, pre-adipocyte and adipocyte 3T3-L1 cells were used to assess the obestatin effect on cell metabolism and adipogenesis based on the regulation of the key enzymatic nodes, Akt and AMPK and their downstream targets. For in vivo assays, white adipose tissue (WAT) was obtained from male rats under continuous subcutaneous infusion of obestatin. Obestatin activated Akt and its downstream targets, GSK3α/β, mTOR and S6K1, in 3T3-L1 adipocyte cells. Simultaneously, obestatin inactivated AMPK in this cell model. In keeping with this, ACC phosphorylation was also decreased. This fact was confirmed in vivo in white adipose tissue (omental, subcutaneous and gonadal) obtained from male rats under continuous sc infusion of obestatin (24 and 72 hrs). The relevance of obestatin as regulator of adipocyte metabolism was supported by AS160 phosphorylation, GLUT4 translocation and augment of glucose uptake in 3T3-L1 adipocyte cells. In contrast, obestatin failed to modify translocation of fatty acid transporters, FATP1, FATP4 and FAT/CD36, to plasma membrane. Obestatin treatment in combination with IBMX and DEX showed to regulate the expression of C/EBPα, C/EBPβ, C/EBPδ and PPARγ promoting adipogenesis. Remarkable, preproghrelin expression, and thus obestatin expression, increased during adipogenesis being sustained throughout terminal differentiation. Neutralization of endogenous obestatin secreted by 3T3-L1 cells by anti-obestatin antibody decreased adipocyte differentiation. Furthermore, knockdown experiments by preproghrelin siRNA supported that obestatin contributes to adipogenesis. In summary, obestatin promotes adipogenesis in an autocrine/paracrine manner, being a regulator of adipocyte metabolism. These data point to a putative role in the pathogenesis of metabolic syndrome.     

MISS-Prot: web server for self/non-self discrimination of protein residue networks in parasites; theory and experiments in Fasciola peptides and Anisakis allergens

Infections caused by human parasites (HPs) affect the poorest 500 million people worldwide but chemotherapy has become expensive, toxic, and/or less effective due to drug resistance. On the other hand, many 3D structures in Protein Data Bank (PDB) remain without function annotation. We need theoretical models to quickly predict biologically relevant Parasite Self Proteins (PSP), which are expressed differentially in a given parasite and are dissimilar to proteins expressed in other parasites and have a high probability to become new vaccines (unique sequence) or drug targets (unique 3D structure). We present herein a model for PSPs in eight different HPs (Ascaris, Entamoeba, Fasciola, Giardia, Leishmania, Plasmodium, Trypanosoma, and Toxoplasma) with 90% accuracy for 15?341 training and validation cases. The model combines protein residue networks, Markov Chain Models (MCM) and Artificial Neural Networks (ANN). The input parameters are the spectral moments of the Markov transition matrix for electrostatic interactions associated with the protein residue complex network calculated with the MARCH-INSIDE software. We implemented this model in a new web-server called MISS-Prot (MARCH-INSIDE Scores for Self-Proteins). MISS-Prot was programmed using PHP/HTML/Python and MARCH-INSIDE routines and is freely available at: . This server is easy to use by non-experts in Bioinformatics who can carry out automatic online upload and prediction with 3D structures deposited at PDB (mode 1). We can also study outcomes of Peptide Mass Fingerprinting (PMFs) and MS/MS for query proteins with unknown 3D structures (mode 2). We illustrated the use of MISS-Prot in experimental and/or theoretical studies of peptides from Fasciola hepatica cathepsin proteases or present on 10 Anisakis simplex allergens (Ani s 1 to Ani s 10). In doing so, we combined electrophoresis (1DE), MALDI-TOF Mass Spectroscopy, and MASCOT to seek sequences, Molecular Mechanics + Molecular Dynamics (MM/MD) to generate 3D structures and MISS-Prot to predict PSP scores. MISS-Prot also allows the prediction of PSP proteins in 16 additional species including parasite hosts, fungi pathogens, disease transmission vectors, and biotechnologically relevant organisms.     

The inhibition of 2-arachidonoyl-glycerol (2-AG) biosynthesis,rather than enhancing striatal damage,protects striatal neurons from malonate-induced death: a potential role of cyclooxygenase-2-dependent metabolism of 2-AG

The cannabinoid CB₂ receptor, which is activated by the endocannabinoid 2-arachidonoyl-glycerol (2-AG), protects striatal neurons from apoptotic death caused by the local administration of malonate, a rat model of Huntington''s disease (HD). In the present study, we investigated whether endocannabinoids provide tonic neuroprotection in this HD model, by examining the effect of O-3841, an inhibitor of diacylglycerol lipases, the enzymes that catalyse 2-AG biosynthesis, and JZL184 or OMDM169, two inhibitors of 2-AG inactivation by monoacylglycerol lipase (MAGL). The inhibitors were injected in rats with the striatum lesioned with malonate, and several biochemical and morphological parameters were measured in this brain area. Similar experiments were also conducted in vitro in cultured M-213 cells, which have the phenotypic characteristics of striatal neurons. O-3841 produced a significant reduction in the striatal levels of 2-AG in animals lesioned with malonate. However, surprisingly, the inhibitor attenuated malonate-induced GABA and BDNF deficiencies and the reduction in Nissl staining, as well as the increase in GFAP immunostaining. In contrast, JZL184 exacerbated malonate-induced striatal damage. Cyclooxygenase-2 (COX-2) was induced in the striatum 24 h after the lesion simultaneously with other pro-inflammatory responses. The COX-2-derived 2-AG metabolite, prostaglandin E₂ glyceryl ester (PGE₂-G), exacerbated neurotoxicity, and this effect was antagonized by the blockade of PGE₂-G action with AGN220675. In M-213 cells exposed to malonate, in which COX-2 was also upregulated, JZL184 worsened neurotoxicity, and this effect was attenuated by the COX-2 inhibitor celecoxib or AGN220675. OMDM169 also worsened neurotoxicity and produced measurable levels of PGE₂-G. In conclusion, the inhibition of 2-AG biosynthesis is neuroprotective in rats lesioned with malonate, possibly through the counteraction of the formation of pro-neuroinflammatory PGE₂-G, formed from COX-2-mediated oxygenation of 2-AG. Accordingly, MAGL inhibition or the administration of PGE₂-G aggravates the malonate toxicity.     

The membranotropic activity of N-terminal peptides from the pore-forming proteins sticholysin I and II is modulated by hydrophobic and electrostatic interactions as well as lipid composition

The sea anemone Stichodactyla helianthus produces two pore-forming proteins, sticholysins I and II (St I and St II). Despite their high identity (93%), these toxins exhibit differences in hemolytic activity that can be related to those found in their N-terminal. To clarify the contribution of the N-terminal amino acid residues to the activity of the toxins, we synthesized peptides spanning residues 1-31 of St I (StI1-31) or 1-30 of St II (StII1-30) and demonstrated that StII1-30 promotes erythrocyte lysis to a higher extent than StI1-31. For a better understanding of the molecular mechanism underlying the peptide activity, here we studied their binding to lipid monolayers and pemeabilizing activity in liposomes. For this, we examined the effect on peptide membranotropic activity of including phospatidic acid and cholesterol in a lipid mixture of phosphatidylcholine and sphingomyelin. The results suggest the importance of continuity of the 1-10 hydrophobic sequence in StII1-30 for displaying higher binding and activity, in spite of both peptides' abilities to form pores in giant unilamellar vesicles. Thus, the different peptide membranotropic action is explained in terms of the differences in hydrophobic and electrostatic peptide properties as well as the enhancing role of membrane inhomogeneities.     

Threading structural model of the manganese-stabilizing protein PsbO reveals presence of two possible beta-sandwich domains.

The manganese-stabilizing protein (PsbO) is an essential component of photosystem II (PSII) and is present in all oxyphotosynthetic organisms. PsbO allows correct water splitting and oxygen evolution by stabilizing the reactions driven by the manganese cluster. Despite its important role, its structure and detailed functional mechanism are still unknown. In this article we propose a structural model based on fold recognition and molecular modeling. This model has additional support from a study of the distribution of characteristics of the PsbO sequence family, such as the distribution of conserved, apolar, tree-determinants, and correlated positions. Our threading results consistently showed PsbO as an all-beta (beta) protein, with two homologous beta domains of approximately 120 amino acids linked by a flexible Proline-Glycine-Glycine (PGG) motif. These features are compatible with a general elongated and flexible architecture, in which the two domains form a sandwich-type structure with Greek key topology. The first domain is predicted to include 8 to 9 beta-strands, the second domain 6 to 7 beta-strands. An Ig-like beta-sandwich structure was selected as a template to build the 3-D model. The second domain has, between the strands, long-loops rich in Pro and Gly that are difficult to model. One of these long loops includes a highly conserved region (between P148 and P174) and a short alpha-helix (between E181 and N188)). These regions are characteristic parts of PsbO and show that the second domain is not so similar to the template. Overall, the model was able to account for much of the experimental data reported by several authors, and it would allow the detection of key residues and regions that are proposed in this article as essential for the structure and function of PsbO.     

Extraction and cleaning methods to detect yessotoxins in contaminated mussels

Yessotoxin (YTX) and its analogues are a newly recognized group of toxins with increased presence in shellfish in recent years. They can be quantified by various functional assays due to their interaction with phosphodiesterases (PDEs). One of these assays detects the binding between the YTX and the fluorescently labeled PDE I using fluorescence polarization, a spectroscopic technique based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. The aim of this study was to develop a YTX extraction procedure from mussels that does not interfere with this detection method. YTX concentrations were measured in spiked mussel extracts obtained through use of different extraction methods and cleaning procedures. The percentages of toxin recovery in various steps of the processes were calculated using these concentrations. Six extraction methods and two cleaning steps were used and no matrix effects and high toxin recoveries were obtained in two cases. One case used acetone as extraction solvent followed by three dichloromethane partitions and the other case used methanol. The cleaning procedure includes a silica cartridge and a 10,000 NMWL filter. Finally these two extraction-cleaning-detection methods were applied to a naturally contaminated mussel sample and results showed that not only YTX but also homoYTX and hydroxyYTX can be quantified with a 85-90% recovery.     

Enhanced production of laccase in <Emphasis Type="Italic">Coriolopsis rigida</Emphasis> grown on barley bran in flask or expanded-bed bioreactor

In this work, the effect on laccase activity of adding xylidine, veratryl alcohol and copper sulphate to cultures of Coriolopsis rigida under submerged cultivation conditions have been investigated. The highest activities (approximately 6 × 10⁵ nkat/l) were obtained when the inducers copper sulphate (2 mM) and xylidine (10 mM) were added simultaneously. In addition, operating in the optimal conditions, it was possible to maintain the sustained production of laccase (around 3 × 10⁵ nkat/l) for successive repeated batch cultures in an expanded-bed laboratory scale bioreactor. On the other hand, in vitro phenol degradation by laccase obtained in the bioreactor was studied with/without an effective mediator 1-hydroxybenzotriazol (HBT). The presence of a radical mediator plays an important role inducing the degradation of phenol, and without mediator the polymerization of phenol was detected.     

Estrogen mediates innate and adaptive immune alterations to influenza infection in pregnant mice

Pregnancy is a leading risk factor for severe complications during an influenza virus infection. Women infected during their second and third trimesters are at increased risk for severe cardiopulmonary complications, premature delivery, and death. Here, we establish a murine model of aerosolized influenza infection during pregnancy. We find significantly altered innate antiviral responses in pregnant mice, including decreased levels of IFN-β, IL-1α, and IFN-γ at early time points of infection. We also find reduced cytotoxic T cell activity and delayed viral clearance. We further demonstrate that pregnancy levels of the estrogen 17-β-estradiol are able to induce key anti-inflammatory phenotypes in immune responses to the virus independently of other hormones or pregnancy-related stressors. We conclude that elevated estrogen levels result in an attenuated anti-viral immune response, and that pregnancy-associated morbidities occur in the context of this anti-inflammatory phenotype.     

Integrative top-down system metabolic modeling in experimental disease states via data-driven Bayesian methods

Multivariate metabolic profiles from biofluids such as urine and plasma are highly indicative of the biological fitness of complex organisms and can be captured analytically in order to derive top-down systems biology models. The application of currently available modeling approaches to human and animal metabolic pathway modeling is problematic because of multicompartmental cellular and tissue exchange of metabolites operating on many time scales. Hence, novel approaches are needed to analyze metabolic data obtained using minimally invasive sampling methods in order to reconstruct the patho-physiological modulations of metabolic interactions that are representative of whole system dynamics. Here, we show that spectroscopically derived metabolic data in experimental liver injury studies (induced by hydrazine and alpha-napthylisothiocyanate treatment) can be used to derive insightful probabilistic graphical models of metabolite dependencies, which we refer to as metabolic interactome maps. Using these, system level mechanistic information on homeostasis can be inferred, and the degree of reversibility of induced lesions can be related to variations in the metabolic network patterns. This approach has wider application in assessment of system level dysfunction in animal or human studies from noninvasive measurements.