Structural and functional characterization of myotoxin, Cr-IV 1, a phospholipase A2 D49 from the venom of the snake Calloselasma rhodostoma

A new D49 PLA(2) was purified from the venom of Calloselasma rhodostoma after two chromatographic steps. Molecular exclusion chromatography was done through a Protein-Pack 300 SW column (0.78 cm x 30 cm), eluting with 0.25 M ammonium bicarbonate, pH 7.9, at a flow rate of 0.3 ml/min. Reverse-phase HPLC was then performed on mu-Bondapack C-18. The sample was determined to have a molecular mass of 13,870.94 Da MALDI-TOF by mass spectrometry, and the amino acid composition showed that Cr-IV 1 presented a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). Cr-IV 1 presented a sequence of 122 amino acid residues: DLWEFGQMILKETGSLPFPY YTTYGCYCGV GGRGGKPKDA TDRCCFVHDC CYGKLTGCPK TNDRYSYSRL DYTIVCGEGG PCKQICECDK AAAVCFRENL RTYNKKYRYHLKPFCKEPAE TC and a calculated pI value of 8.0. Cr-IV 1 had PLA(2) activity in the presence of a synthetic chromogenic substrate (4-nitro-3-(octanoyloxy)benzoic acid) and showed a rapid cytolytic effect on mouse skeletal muscle myoblasts and myotubes in culture. In mice, Cr-IV 1 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD(50) of Cr-IV 1 was determined to be 0.07 mg/k body weight by intracerebroventricular (i.c.v.) injection. The combination of structural and functional information obtained herein classifies Cr-IV 1 as a new member of the D49 PLA(2) family, as it presents the typical behavior of a phospholipase A(2) from this family.     

Genotoxic damage in Solea senegalensis exposed to sediments from the Sado Estuary (Portugal): effects of metallic and organic contaminants

Juvenile Solea senegalensis (Senegalese sole) were exposed to freshly collected sediments from three sites of the Sado Estuary (West-Portuguese coast) in 28-day laboratory assays in order to assess the ecological risk from sediment contaminants, by measuring two genotoxicity biomarkers in peripheral blood: the percentage of Erythrocyte Nuclear Abnormalities (ENA) by use of an adaptation of the micronucleus test, and the percentage of DNA strand-breakage (DNA-SB) with the Comet assay. Sediments were surveyed for metallic (Cr, Ni, Cu, Zn, As, Cd and Pb) and organic (PAHs (polycyclic aromatic hydrocarbons), PCBs (polychlorinated biphenyls) and DDTs (dichloro-diphenyl-trichloroethane)) contaminants. Sediments from site A (farthest from hotspots of contamination) were found to be the least contaminated and weaker inducers of genotoxic damage, whereas sediments from sites B (urban influence) and C (affected by industrial effluents and agricultural runoffs) were responsible for a very significant increase in both ENA and DNA-SB, site B being most contaminated with metals and site C mainly with organic pollutants, especially PAHs and PCBs . Analysis of genotoxic effects showed a strong correlation between the concentrations of PAHs and PCBs and both biomarkers at sampling times T(14) and T(28), while the amounts of Cu, As, Cd and Pb were less strongly correlated, and at T(28) only, with ENA and DNA-SB. These results show that organic contaminants in sediment are stronger and faster acting genotoxic stressors. The results also suggest that metals may have an inhibitory effect on genotoxicity when interacting with organic contaminants, at least during early exposure. ENA and DNA-SB do not show a linear relationship, but a strong correlation exists between the overall increase in genotoxicity caused by exposure to sediment, confirming that they are different, and possibly non-linked effects that respond similarly to exposure. Although the Comet assay showed enhanced sensitivity, the two analyses are complementary and suitable for the biomonitoring of sediment contaminants in a benthic species like S. senegalensis.     

Binding of phospholipids to β-Lactoglobulin and their transfer to lipid bilayers

The bovine milk lipocalin, β-Lactoglobulin (β-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC₁₀PE) to β-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, K_B, was (1.2 ± 0.2) × 10⁶ M^− 1 at 25 °C, pH 7.4 and I = 0.15 M. Dependence of K_B on pH and on the monomer-dimer equilibrium of β-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the β-barrel in the protein. The monomer-dimer equilibrium of β-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, K_D, was (7.0 ± 1.5) × 10⁵ M^− 1 at 25 °C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC₁₀PE between β-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. β-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.     

Chemical Composition,Antibacterial, Antibiofilm and Synergistic Properties of Essential Oils from Eucalyptus globulus Labill. and Seven Mediterranean Aromatic Plants

Essential oils (EOs) from Eucalyptus globulus Labill . ssp. globulus and from Mediterranean autochthonous aromatic plants – Thymus mastichina L., Mentha pulegium L., Rosmarinus officinalis L., Calamintha nepeta (L.) Savi ssp. nepeta, Cistus ladanifer L., Foeniculum vulgare L., Dittrichia viscosa (L.) Greuter ssp. viscosa – were extracted by hydrodistillation and characterized by GC‐FID and NMR spectroscopy. EOs were evaluated for antimicrobial properties against several bacterial strains, using diverse methods, namely, the agar disc‐diffusion method, the microdilution method, the crystal violet assay and the Live/Dead staining for assessment of biofilm formation. Potential synergy was assessed by a checkerboard method. EOs of R. officinalis and C. ladanifer showed a predominance in monoterpene hydrocarbons (> 60%); EOs of C. nepeta, M. pulegium, T. mastichina, E. globulus and F. vulgare were rich in oxygenated monoterpenes (62 – 96%) whereas EO of D. viscosa was mainly composed of oxygenated sesquiterpenes (54%). All EOs showed antimicrobial activity; M. pulegium and E. globulus generally had the strongest antimicrobial activity. EO of C. nepeta was the most promising in hampering the biofilm formation. The combinations D. viscosa/C. nepeta and E. globulus/T. mastichina were synergistic against Staphylococcus aureus. These results support the notion that EOs from the aromatic plants herein reported should be further explored as potential pharmaceuticals and/or food preservatives.     

Analysis of muscle activation during different leg press exercises at submaximum effort levels

Many studies have analyzed muscle activity during different strength exercises. Although the leg press (LP) is one of the most common exercises performed, there is little evidence of lower limb muscle activity patterns during this exercise and its variations. Thus, this study aimed to verify how mechanical changes and loads affect lower limb muscle activity during the performance of different LP exercises. Fourteen women performed 3 LP exercises: 45 degrees LP (LP45), LP high (LPH), and LP low (LPL) at 40% and 80% of the 1 repetition maximum. The electromyographic activity of the rectus femoris, vastus lateralis, biceps femoris, gastrocnemius, and gluteus maximus was recorded. Results suggested that mechanical changes affect lower limb muscle activity and that it is related to the load used. At moderate effort levels, the rectus femoris and gastrocnemius were more active during the LP45 and LPL than during the LPH. At a high effort level, the rectus femoris and vastus lateralis (quadriceps) were more active during the LPL than the LPH. Again, the rectus femoris and gastrocnemius were more active during the LP45 and LPL than the LPH. On the other hand, gluteus maximus activity was greater during the LPH than the LPL. This study found that coordination patterns of muscle activity are different when performing LP variations at high or moderate effort levels because of mechanical changes and different loads lifted during the different LP exercises. These results suggest that if the goal is to induce greater rectus femoris and vastus lateralis (quadriceps) activation, the LPL should be performed. On the other hand, if the goal is to induce gluteus maximus activity, the LPH should be performed.     

Lipids revert inert Abeta amyloid fibrils to neurotoxic protofibrils that affect learning in mice

Although soluble oligomeric and protofibrillar assemblies of Abeta-amyloid peptide cause synaptotoxicity and potentially contribute to Alzheimer's disease (AD), the role of mature Abeta-fibrils in the amyloid plaques remains controversial. A widely held view in the field suggests that the fibrillization reaction proceeds 'forward' in a near-irreversible manner from the monomeric Abeta peptide through toxic protofibrillar intermediates, which subsequently mature into biologically inert amyloid fibrils that are found in plaques. Here, we show that natural lipids destabilize and rapidly resolubilize mature Abeta amyloid fibers. Interestingly, the equilibrium is not reversed toward monomeric Abeta but rather toward soluble amyloid protofibrils. We characterized these 'backward' Abeta protofibrils generated from mature Abeta fibers and compared them with previously identified 'forward' Abeta protofibrils obtained from the aggregation of fresh Abeta monomers. We find that backward protofibrils are biochemically and biophysically very similar to forward protofibrils: they consist of a wide range of molecular masses, are toxic to primary neurons and cause memory impairment and tau phosphorylation in mouse. In addition, they diffuse rapidly through the brain into areas relevant to AD. Our findings imply that amyloid plaques are potentially major sources of soluble toxic Abeta-aggregates that could readily be activated by exposure to biological lipids.     

Reassessment of the vocal repertoire of a nest-building gladiator frog,Boana pardalis (Anura,Hylidae, Cophomantinae): implications for its diagnosis within the B. faber species group

In this study, we re-describe the advertisement and territorial calls of Boana pardalis, carry out an acoustic comparison between the studied species and the other congeners of the B. faber group, and report for the first time the tympanic amplexus for the studied species. The advertisement call of B. pardalis can be used to supplement its diagnosis in the B. faber group based on temporal call traits, e.g. emission rate and emission pattern, as well as the call envelope.     

Protective effect of mannan oligosaccharides against early colonization by Salmonella Enteritidis in chicks is improved by higher dietary threonine levels

Aims

To evaluate mannan oligosaccharide (MOS) and threonine effects on performance, small intestine morphology and Salmonella spp. counts in Salmonella Enteritidis‐challenged birds.

Methods and Results

One‐day‐old chicks (1d) were distributed into five treatments: nonchallenged animals fed basal diet (RB‐0), animals fed basal diet and infected with Salmonella Enteritidis (RB‐I), animals fed high level of threonine and infected (HT‐I), birds fed basal diet with MOS and infected (MOS‐I), birds fed high level of threonine and MOS and infected (HT+MOS‐I). Birds were inoculated at 2d with Salmonella Enteritidis, except RB‐0 birds. Chicks fed higher dietary threonine and MOS showed performance similar to RB‐0 and intestinal morphology recovery at 8 dpi. Salmonella counts and the number of Salmonella‐positive animals were lower in HT+MOS‐I compared with other challenged groups.

Conclusion

Mannan oligosaccharides and threonine act synergistically, resulting in improved intestinal environment and recovery after Salmonella inoculation.

Significance and Impact of the Study

Nutritional approaches may be useful to prevent Salmonella infection in the first week and putative carcass contamination at slaughter. This is the first report on the possible synergistic effect of mannan oligosaccharides and threonine, and further studies should be performed including performance, microbiota evaluation, composition of intestinal mucins and immune assessment.     

Laminin‐γ1 chain and stress inducible protein 1 synergistically mediate PrPC‐dependent axonal growth via Ca2+ mobilization in dorsal root ganglia neurons

Prion protein (PrP^C) is a cell surface glycoprotein that is abundantly expressed in nervous system. The elucidation of the PrP^C interactome network and its significance on neural physiology is crucial to understanding neurodegenerative events associated with prion and Alzheimer's diseases. PrP^C co‐opts stress inducible protein 1/alpha7 nicotinic acetylcholine receptor (STI1/α7nAChR) or laminin/Type I metabotropic glutamate receptors (mGluR1/5) to modulate hippocampal neuronal survival and differentiation. However, potential cross‐talk between these protein complexes and their role in peripheral neurons has never been addressed. To explore this issue, we investigated PrP^C‐mediated axonogenesis in peripheral neurons in response to STI1 and laminin‐γ1 chain‐derived peptide (Ln‐γ1). STI1 and Ln‐γ1 promoted robust axonogenesis in wild‐type neurons, whereas no effect was observed in neurons from PrP^C‐null mice. PrP^C binding to Ln‐γ1 or STI1 led to an increase in intracellular Ca²⁺ levels via distinct mechanisms: STI1 promoted extracellular Ca²⁺ influx, and Ln‐γ1 released calcium from intracellular stores. Both effects depend on phospholipase C activation, which is modulated by mGluR1/5 for Ln‐γ1, but depends on, C‐type transient receptor potential (TRPC) channels rather than α7nAChR for STI1. Treatment of neurons with suboptimal concentrations of both ligands led to synergistic actions on PrP^C‐mediated calcium response and axonogenesis. This effect was likely mediated by simultaneous binding of the two ligands to PrP^C. These results suggest a role for PrP^C as an organizer of diverse multiprotein complexes, triggering specific signaling pathways and promoting axonogenesis in the peripheral nervous system.