Curcumin derivatives as new ligands of Aβ peptides

Curcumin derivatives with high chemical stability, improved solubility and carrying a functionalized appendage for the linkage to other entities, have been synthesized in a straightforward manner. All compounds retained Curcumin ability to bind Aβ peptide oligomers without inducing their aggregation. Moreover all Curcumin derivatives were able to stain very efficiently Aβ deposits.     

Unraveling the molecular basis for ligand binding in truncated hemoglobins: The trHbO Bacillus subtilis case

Truncated hemoglobins (trHbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. Their tertiary structure consists in a 2‐over‐2 helical sandwich, which display typically an inner tunnel/cavity system for ligand migration and/or storage. The microorganism Bacillus subtilis contains a peculiar trHb, which does not show an evident tunnel/cavity system connecting the protein active site with the solvent, and exhibits anyway a very high oxygen association rate. Moreover, resonant Raman results of CO bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. To understand these experimental results with atomistic detail, we performed classical molecular dynamics simulations of the oxy, carboxy, and deoxy proteins. The free energy profiles for ligand migration suggest that there is a key residue, GlnE11, that presents an alternate conformation, in which a wide ligand migration tunnel is formed, consistently with the kinetic data. This tunnel is topologically related to the one found in group I trHbs. On the other hand, the results for the CO and O₂ bound protein show that GlnE11 is directly involved in the stabilization of the cordinated ligand, playing a similar role as TyrB10 and TrpG8 in other trHbs. Our results not only reconcile the structural data with the kinetic information, but also provide additional insight into the general behaviour of trHbs. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Formation of methionine sulfoxide by peroxynitrite at position 1606 of von Willebrand factor inhibits its cleavage by ADAMTS-13: A new prothrombotic mechanism in diseases associated with oxidative stress

An enhanced formation of reactive oxygen species and peroxynitrite occurs in several clinical settings including diabetes, coronary artery disease, stroke, sepsis, and chronic inflammatory diseases. Peroxynitrite oxidizes methionine and tyrosine residues to methionine sulfoxide (MetSO) and 3-nitrotyrosine (NT), respectively. Notably, ADAMTS-13 cleaves von Willebrand factor (VWF) exclusively at the Tyr1605–Met1606 peptide bond in the A2 domain. We hypothesized that peroxynitrite could oxidize either or both of these amino acid residues, thus potentially affecting ADAMTS-13-mediated cleavage. We tested our hypothesis using synthetic peptide substrates based on: (1) VWF Asp1596–Ala1669 sequence (VWF74) and (2) VWF Asp1596–Ala1669 sequence containing nitrotyrosine (VWF74-NT) or methionine sulfoxide (VWF74-MetSO) at position 1605 or 1606, respectively. The peptides were treated with recombinant ADAMTS-13 and the cleavage products analyzed by RP-HPLC. VWF74 oxidized by peroxynitrite underwent a severe impairment of its hydrolysis. Likewise, VWF74-MetSO was minimally hydrolyzed, whereas VWF74-NT was hydrolyzed slightly more efficiently than VWF74. Oxidation by peroxynitrite of purified VWF multimers inhibited ADAMTS-13 hydrolysis, but did not alter their electrophoretic pattern nor their ability to induce platelet agglutination by ristocetin. Moreover, VWF purified from type 2 diabetic patients showed oxidative damage, as revealed by enhanced carbonyl, NT, and MetSO content and was partially resistant to ADAMTS-13 hydrolysis. In conclusion, peroxynitrite may contribute to prothrombotic effects, hindering the proteolytic processing by ADAMTS-13 of high-molecular-weight VWF multimers, which have the highest ability to bind and activate platelets in the microcirculation.     

Expression of pro-inflammatory TACE-TNF-α-amphiregulin axis in Sjögren’s syndrome salivary glands

The tumor-necrosis-factor-converting-enzyme (TACE)-TNF-α-Amphiregulin (AREG) axis plays an important pathogenic role in inflammatory and autoimmune disorders. However, the pathological roles of these proteins in the chronic autoimmune disease Sjögren’s syndrome (SS) remain to be elucidated. It is known that the TACE–AREG axis is clearly part of a larger cascade of signals that starts with the activation of Furin, responsible for maturation of TACE that, in turn, determines the production of active TNF-α, directly involved in the up-regulation of AREG expression. This study showed that Furin, TACE, TNF-α, and AREG proteins, detected in acinar and ductal cells of human salivary glands from SS patients, increased remarkably in comparison with biopsies of labial salivary glands from healthy controls. The changes in Furin, TACE, TNF- α, and AREG proteins’ level detected in salivary glands biopsies of SS patients could be responsible for pro-inflammatory cytokines overexpression characterizing Sjögren’s syndrome.     

Modelling of the provisional side-branch stenting approach for the treatment of atherosclerotic coronary bifurcations: effects of stent positioning

The most common approach to treat atherosclerosis in coronary bifurcations is the provisional side-branch (PSB) stenting, which consists sequentially of the insertion of a stent in the main branch (MB) of the bifurcation and a dilatation of the side branch (SB) passing through the struts of the stent at the bifurcation. This approach can be followed by a redilatation of the MB only or by a Final Kissing Balloon (FKB) inflation, both strategies leading to a minor stent distortion in the MB. The positioning of the stent struts in the bifurcation and the stresses generated in the stent and vessel wall are worthy of investigation for a better understanding of the mechanobiology of the system. For this purpose, a computer model of an atherosclerotic coronary bifurcation based on the finite element method was developed; the effects of performing the final redilatation with the two strategies utilising one or two balloons and those created by a different stent strut positioning around the SB were investigated. Results correlate well with previous experimental tests regarding the deformation following balloon expansion. Furthermore, results confirm firstly that the re-establishment of an optimal spatial configuration of the stent after the PSB approach is achieved with both strategies; secondly, results show that case of stent positioning with one cell placed centrally (with regard to the SB) should be preferred, avoiding the presence of struts inside the vessel lumen, which may reduce hemodynamic disturbances. The central positioning also resulted in a better solution in terms of lower stresses in the stent struts and, more importantly, in the vascular tissues.     

Single-molecule force spectroscopy of cartilage aggrecan self-adhesion

We investigated self-adhesion between highly negatively charged aggrecan macromolecules extracted from bovine cartilage extracellular matrix by performing atomic force microscopy (AFM) imaging and single-molecule force spectroscopy (SMFS) in saline solutions. By controlling the density of aggrecan molecules on both the gold substrate and the gold-coated tip surface at submonolayer densities, we were able to detect and quantify the Ca²⁺-dependent homodimeric interaction between individual aggrecan molecules at the single-molecule level. We found a typical nonlinear sawtooth profile in the AFM force-versus-distance curves with a molecular persistence length of l_p = 0.31 ± 0.04 nm. This is attributed to the stepwise dissociation of individual glycosaminoglycan (GAG) side chains in aggrecans, which is very similar to the known force fingerprints of other cell adhesion proteoglycan systems. After studying the GAG-GAG dissociation in a dynamic, loading-rate-dependent manner (dynamic SMFS) and analyzing the data according to the stochastic Bell-Evans model for a thermally activated decay of a metastable state under an external force, we estimated for the single glycan interaction a mean lifetime of τ = 7.9 ± 4.9 s and a reaction bond length of x_β = 0.31 ± 0.08 nm. Whereas the x_β-value compares well with values from other cell adhesion carbohydrate recognition motifs in evolutionary distant marine sponge proteoglycans, the rather short GAG interaction lifetime reflects high intermolecular dynamics within aggrecan complexes, which may be relevant for the viscoelastic properties of cartilage tissue.     

Effect of n-3 and n-6 polyunsaturated fatty acids on hare reproductive performances

The study was carried out on 42 breeder couples (42 males and 42 females) of European brown hare (Lepus europaeus), divided into three groups fed three different experimental diets (14 couples/treatment). Two diets were supplemented with n-3 and n-6 polyunsaturated fatty acids (PUFAs; 2% of linseed oil and soybean oil, respectively) and were compared with a control diet supplemented with a monounsaturated fatty acids (2% of olive oil). During the experimental period (from 15 April to 30 September), the following parameters were recorded: days from the beginning of trial to the first parturition, parturition interval, number of parturitions, number of leverets born (alive and dead), dead during suckling, the total number of leverets weaned and feed intake per cage (of males, females and leverets until weaning). Feed intake was not influenced by treatments. In hares fed n-3 and n-6 diets, the days from the beginning of the trial to the first parturition and the parturition interval were similar and were lower compared with control group (63.1 v. 70.6 days, and 37.8 v. 40.9 days, respectively; P < 0.05). Hares from n-6 group had a higher (P < 0.05) number of parturitions per cage during the experimental period than the n-3 and control group that showed a similar value (3.00 v. 2.36, respectively). The total number of leverets born per cage and parturition in n-6 and n-3 groups increased with respect to those fed control diet (P < 0.05). The leverets' mortality rate at birth was higher in n-6 than in n-3 and control group (3.50 v. 2.17, respectively; P < 0.05). In control group, leverets' mortality rate during suckling was lower with respect to n-3 (P < 0.05) and n-6 (P < 0.05), showing the highest value for the latter (P < 0.05). In spite of this higher mortality, the number of leverets weaned per cage and parturition was higher (P < 0.05) in n-6 compared with n-3 group, being the latter higher than the control group (3.12, 2.79 and 2.43, respectively). Our results show that the dietary PUFAs, particularly n-6 supplementation, have a positive influence on the reproductive performances of the European brown hare.     

SYBR green real-time PCR method to detect Clostridium botulinum type A

Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 x 10(1) copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%).     

Sentinels at the wall: cell wall receptors and sensors

The emerging view of the plant cell wall is of a dynamic and responsive structure that exists as part of a continuum with the plasma membrane and cytoskeleton. This continuum must be responsive and adaptable to normal processes of growth as well as to stresses such as wounding, attack from pathogens and mechanical stimuli. Cell expansion involving wall loosening, deposition of new materials, and subsequent rigidification must be tightly regulated to allow the maintenance of cell wall integrity and co-ordination of development. Similarly, sensing and feedback are necessary for the plant to respond to mechanical stress or pathogen attack. Currently, understanding of the sensing and feedback mechanisms utilized by plants to regulate these processes is limited, although we can learn from yeast, where the signalling pathways have been more clearly defined. Plant cell walls possess a unique and complicated structure, but it is the protein components of the wall that are likely to play a crucial role at the forefront of perception, and these are likely to include a variety of sensor and receptor systems. Recent plant research has yielded a number of interesting candidates for cell wall sensors and receptors, and we are beginning to understand the role that they may play in this crucial aspect of plant biology.     

Effects of parity and age on female attraction to faces of infants and neonates in rhesus macaques

This study investigated the effects of parity and age on female rhesus macaque attention toward infants, and assessed whether the faces of neonates are more attractive than those of older infants. Six nulliparous and six multiparous females were shown digitized images of neonates’ and 5- to 6-month-old infants’ faces. Attention and preferences for images were measured by gaze duration and other picture-directed behaviors, including lip smacking, approaches, and presentations. As predicted, nulliparous females displayed significantly longer gaze durations for images than did multiparous females. There were no significant differences in gaze duration for faces of neonates and those of infants, but images of infants were approached more frequently than images of neonates. This difference is tentatively explained on the basis of differences in female familiarity with neonates’ and infants’ faces and differences in opportunities for allomothering with neonates and infants.