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.     

Heterogeneous mechanomyographic absolute activation of paraspinal muscles assessed by a two-dimensional array during short and sustained contractions

Spatial dependency of paraspinal muscle activity was assessed using a new two-dimensional MMG recording system. MMG signals were detected over the left and right paraspinal muscles of 10 volunteers using a grid of 12 accelerometers. During two separate trials subjects maintained a 20 degrees flexed position and held loads that ranged from 0 to 15 kg (in 2.5 kg increments) for 20s; and 7.5 kg for 6 min. Maps of absolute and normalised (with respect to initial values) average rectified value, mean power frequency, variance and skewness of the power spectral density were obtained from the two-dimensional MMG recordings. For both the short duration and sustained contractions, the MMG absolute average rectified value, mean power frequency, variance and skewness depended on accelerometer location (P<0.05), while, with the exception of the skewness (P<0.05), normalised values did not. These results demonstrate both inhomogeneous MMG absolute activity and homogeneous MMG normalised activity in paraspinal muscles for short duration and sustained contractions. Moreover, the effect of accelerometer location on spectral variables confirmed the limited validity of general relationships between MMG spectral changes and motor unit recruitment strategies. This study underlines the importance of using multiple recording sites when assessing back muscle activity.     

Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress

Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordon's hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane-coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress.     

Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2

Biogenesis and recycling of synaptic vesicles are accompanied by sorting processes that preserve the molecular composition of the compartments involved. In the present study, we have addressed the targeting of synaptobrevin 2/VAMP2 (vesicle-associated membrane protein 2), a critical component of the synaptic vesicle--fusion machinery, in a heterotypic context where its sorting is not confounded by the presence of other neuron-specific molecules. Ectopically expressed synaptophysin I interacts with VAMP2 and alters its default surface targeting to a prominent vesicular distribution, with no effect on the targeting of other membrane proteins. Protein-protein interaction is not sufficient for the control of VAMP2 sorting, which is mediated by the C-terminal domain of synaptophysin I. Synaptophysin I directs the sorting of VAMP2 to vesicles before surface delivery, without influencing VAMP2 endocytosis. Consistent with this, dynamin and alpha-SNAP (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein) mutants which block trafficking at the plasma membrane do not abrogate the effect of synaptophysin I on VAMP2 sorting. These results indicate that the sorting determinants of synaptic vesicle proteins can operate independently of a neuronal context and implicate the association of VAMP2 with synaptophysin I in the specification of the pathway of synaptic vesicle biogenesis.     

An experimental and theoretical study of the morphine binding capacity and kinetics of an engineered opioid receptor

Electrochemical real-time monitoring of ligand binding to an engineered opioid receptor specific for morphine is reported. In the particular systems studied, 90% of the binding was found to be completed after only 85-120 s. Thus, the binding kinetics has proven to be more rapid than previously believed. The observed association rate constant for the morphine binding reaction was calculated to be 215 M(-1)s(-1). A theoretical analysis of the experimental binding data suggested that the binding sites of the engineered opioid receptor could best be described by a model having two populations of binding sites: K(D)=40 microM (13 micromol/g) and K(D)=205 microM (29 micromol/g). Furthermore, a theoretical model was developed in order to explain the observed binding of the engineered opioid receptor. This model suggested that the binding sites on the polymer surface are up to 5.1A deep and they allow 100% of the ligand (morphine) to anchor itself into the site. The predicted theoretical maximum binding capacity for the reported receptor is calculated to be approximately 2 mmol/g polymer (based on an increase of cavity density).     

Time-resolved methods in biophysics. 4. Broadband pump-probe spectroscopy system with sub-20 fs temporal resolution for the study of energy transfer processes in photosynthesis.

In this paper we discuss how to push the temporal resolution limits of transient absorption spectroscopy in order to detect very fast processes (energy relaxation, energy or charge transfer, vibrational coherence) taking place in molecules of biological relevance. After reviewing the main principles of femtosecond pump-probe spectroscopy, we describe an experimental setup based on two synchronized non-collinear optical parametric amplifiers (NOPAs). Each NOPA can be independently configured to generate ultra-broadband sub-10 fs visible pulses, tunable 10-15 fs visible pulses, tunable 15-40 fs near-infrared pulses (900-1500 nm). This system enables to perform pump-probe experiments over nearly two octaves of spectrum with sub-20 fs temporal resolution. We then present an application example highlighting the capability of this instrument to track excited state dynamics in biomolecules on the sub-100 fs timescale: the study of carotenoid-bacteriochlorophyll energy transfer processes in peripheral light-harvesting complexes (LH2) from purple bacteria. We show that, by comparing excited-state dynamics of the carotenoids in organic solvents and inside the LH2 complexes, it is possible to visualize in the time domain the primary events in photosynthesis.