Homogentisate 1,2 dioxygenase is expressed in human osteoarticular cells: implications in alkaptonuria

Alkaptonuria (AKU) results from defective homogentisate1,2-dioxygenase (HGD), causing degenerative arthropathy. The deposition of ochronotic pigment in joints is so far attributed to homogentisic acid produced by the liver, circulating in the blood and accumulating locally. Human normal and AKU osteoarticular cells were tested for HGD gene expression by RT-PCR, mono- and 2D-Western blotting. HGD gene expression was revealed in chondrocytes, synoviocytes, osteoblasts. Furthermore, HGD expression was confirmed by Western blotting, that also revealed the presence of five enzymatic molecular species. Our findings indicate that AKU osteoarticular cells produce the ochronotic pigment in loco and this may strongly contribute to induction of ochronotic arthropathy.     

A simple strategy for detecting moving objects during locomotion revealed by animal-robot interactions

An important role of visual systems is to detect nearby predators, prey, and potential mates, which may be distinguished in part by their motion. When an animal is at rest, an object moving in any direction may easily be detected by motion-sensitive visual circuits. During locomotion, however, this strategy is compromised because the observer must detect a moving object within the pattern of optic flow created by its own motion through the stationary background. However, objects that move creating back-to-front (regressive) motion may be unambiguously distinguished from stationary objects because forward locomotion creates only front-to-back (progressive) optic flow. Thus, moving animals should exhibit an enhanced sensitivity to regressively moving objects. We explicitly tested this hypothesis by constructing a simple fly-sized robot that was programmed to interact with a real fly. Our measurements indicate that whereas walking female flies freeze in response to a regressively moving object, they ignore a progressively moving one. Regressive motion salience also explains observations of behaviors exhibited by pairs of walking flies. Because the assumptions underlying the regressive motion salience hypothesis are general, we suspect that the behavior we have observed in Drosophila may be widespread among eyed, motile organisms.     

Fine tuning of the active site modulates specificity in the interaction of O-acetylserine sulfhydrylase isozymes with serine acetyltransferase

O-acetylserine sulfhydrylase (OASS) catalyzes the synthesis of l-cysteine in the last step of the reductive sulfate assimilation pathway in microorganisms. Its activity is inhibited by the interaction with serine acetyltransferase (SAT), the preceding enzyme in the metabolic pathway. Inhibition is exerted by the insertion of SAT C-terminal peptide into the OASS active site. This action is effective only on the A isozyme, the prevalent form in enteric bacteria under aerobic conditions, but not on the B-isozyme, the form expressed under anaerobic conditions. We have investigated the active site determinants that modulate the interaction specificity by comparing the binding affinity of thirteen pentapeptides, derived from the C-terminal sequences of SAT of the closely related species Haemophilus influenzae and Salmonella typhimurium, towards the corresponding OASS-A, and towards S. typhimurium OASS-B. We have found that subtle changes in protein active sites have profound effects on protein–peptide recognition. Furthermore, affinity is strongly dependent on the pentapeptide sequence, signaling the relevance of P3–P4–P5 for the strength of binding, and P1–P2 mainly for specificity. The presence of an aromatic residue at P3 results in high affinity peptides with K_diss in the micromolar and submicromolar range, regardless of the species. An acidic residue, like aspartate at P4, further strengthens the interaction and results in the higher affinity ligand of S. typhimurium OASS-A described to date. Since OASS knocked-out bacteria exhibit a significantly decreased fitness, this investigation provides key information for the development of selective OASS inhibitors, potentially useful as novel antibiotic agents.     

Consistency and Diversity of Spike Dynamics in the Neurons of Bed Nucleus of Stria Terminalis of the Rat: A Dynamic Clamp Study

Neurons display a high degree of variability and diversity in the expression and regulation of their voltage-dependent ionic channels. Under low level of synaptic background a number of physiologically distinct cell types can be identified in most brain areas that display different responses to standard forms of intracellular current stimulation. Nevertheless, it is not well understood how biophysically different neurons process synaptic inputs in natural conditions, i.e., when experiencing intense synaptic bombardment in vivo. While distinct cell types might process synaptic inputs into different patterns of action potentials representing specific “motifs” of network activity, standard methods of electrophysiology are not well suited to resolve such questions. In the current paper we performed dynamic clamp experiments with simulated synaptic inputs that were presented to three types of neurons in the juxtacapsular bed nucleus of stria terminalis (jcBNST) of the rat. Our analysis on the temporal structure of firing showed that the three types of jcBNST neurons did not produce qualitatively different spike responses under identical patterns of input. However, we observed consistent, cell type dependent variations in the fine structure of firing, at the level of single spikes. At the millisecond resolution structure of firing we found high degree of diversity across the entire spectrum of neurons irrespective of their type. Additionally, we identified a new cell type with intrinsic oscillatory properties that produced a rhythmic and regular firing under synaptic stimulation that distinguishes it from the previously described jcBNST cell types. Our findings suggest a sophisticated, cell type dependent regulation of spike dynamics of neurons when experiencing a complex synaptic background. The high degree of their dynamical diversity has implications to their cooperative dynamics and synchronization.     

Quantitative underwater 3D motion analysis using submerged video cameras: accuracy analysis and trajectory reconstruction

In this study we aim at investigating the applicability of underwater 3D motion capture based on submerged video cameras in terms of 3D accuracy analysis and trajectory reconstruction. Static points with classical direct linear transform (DLT) solution, a moving wand with bundle adjustment and a moving 2D plate with Zhang's method were considered for camera calibration. As an example of the final application, we reconstructed the hand motion trajectories in different swimming styles and qualitatively compared this with Maglischo's model. Four highly trained male swimmers performed butterfly, breaststroke and freestyle tasks. The middle fingertip trajectories of both hands in the underwater phase were considered. The accuracy (mean absolute error) of the two calibration approaches (wand: 0.96 mm – 2D plate: 0.73 mm) was comparable to out of water results and highly superior to the classical DLT results (9.74 mm). Among all the swimmers, the hands' trajectories of the expert swimmer in the style were almost symmetric and in good agreement with Maglischo's model. The kinematic results highlight symmetry or asymmetry between the two hand sides, intra- and inter-subject variability in terms of the motion patterns and agreement or disagreement with the model. The two outcomes, calibration results and trajectory reconstruction, both move towards the quantitative 3D underwater motion analysis.     

Growth characteristics of Arthrospira platensis cultured inside a new close-coil photobioreactor incorporating a mandrel to control culture temperature

The aim of this study was to investigate Arthrospira growth inside a new CCP incorporating a mandrel for culture temperature control. Some hydrodynamic aspects and photobioreactor performances were investigated as well. The bioreactor incorporated A. platensis grown under batch and semicontinuous conditions. Two systems were used to recycle Arthrospira cultures: a peristaltic pump and an airlift system. When the pump recycled the culture, we achieved a very high Dean number (De=3,950), which decreased a great deal when the pump was replaced with the airlift system. During outdoor Arthrospira batch growth, a cell concentration of 16.4 g (DW)l-1 was reached after 9 days. However, the maximum chlorophyll content of the biomass (2.0% of DW) was achieved on the fifth and sixth days. The highest daily biomass output rate was obtained using the airlift system, when the CCP was operated under a semicontinuous regime: the gross output rate was 2.85+/-0.37 g (DW) l-1 d-1 and the net was 2.32+/-0.11 g (DW) l-1 d-1. The advantages of the airlift system may be due to the low concentration of oxygen built up inside Arthrospira culture and the lack of cell damage due to the pump system. Thus, oxygen and pump stress may have been avoided.     

Earliest evidence of a primitive cultivar of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. during the Bronze Age in Sardinia (Italy)

The discovery of the Nuragic culture settlement of Sa Osa, Cabras-Oristano, Sardinia, has made it possible to investigate the domestication status of waterlogged uncharred grape pips that were recovered from three wells dating from the Middle and Late Bronze Age (ca. 1350–1150 bc). Applying the stepwise linear discriminant analysis method, a morphological comparison of archaeological seeds and modern wild and cultivated Sardinian grapes pips was performed to determine the similarities between them. The results showed that the archaeological seeds from the Middle Bronze Age have intermediate morphological traits between modern wild and cultivated grape pips from Sardinia. In contrast, the analyses performed on the archaeological seeds from the Late Bronze Age showed a high degree of similarity with the modern cultivars in Sardinia. These results provide the first evidence of primitive cultivated Vitis vinifera in Sardinia during the Late Bronze Age (1286–1115 cal bc, 2σ). This evidence may support the hypothesis that Sardinia could have been a secondary domestication centre of the grapevine, due to the presence of ancient cultivars that still exhibit the phenotypic characteristics of wild grapes.     

Molecular Characterization of the Multiple Interactions of SpsD,a Surface Protein from Staphylococcus pseudintermedius,with Host Extracellular Matrix Proteins

Staphylococcus pseudintermedius, a commensal and pathogen of dogs and occasionally of humans, expresses surface proteins potentially involved in host colonization and pathogenesis. Here, we describe the cloning and characterization of SpsD, a surface protein of S. pseudintermedius reported as interacting with extracellular matrix proteins and corneocytes. A ligand screen and Western immunoblotting revealed that the N-terminal A domain of SpsD bound fibrinogen, fibronectin, elastin and cytokeratin 10. SpsD also interfered with thrombin-induced fibrinogen coagulation and blocked ADP-induced platelet aggregation. The binding site for SpsD was mapped to residues 395–411 in the fibrinogen γ-chain, while binding sites in fibronectin were localized to the N- and C-terminal regions. SpsD also bound to glycine- and serine-rich omega loops within the C-terminal tail region of cytokeratin 10. Ligand binding studies using SpsD variants lacking the C-terminal segment or containing an amino-acid substitution in the putative ligand binding site provided insights into interaction mechanism of SpsD with the different ligands. Together these data demonstrate the multi-ligand binding properties of SpsD and illustrate some interesting differences in the variety of ligands bound by SpsD and related proteins from S. aureus.