Computer Simulation of Assembly and Co-operativity of Hexameric AAA ATPases

AAA ATPases form a functionally diverse superfamily of proteins. Most members form homo-hexameric ring complexes, are catalytically active only in the fully assembled state, and show co-operativity among the six subunits. The mutual dependence among the subunits is clearly evidenced by the fact that incorporation of mutated, inactive subunits can decrease the activity of the remaining wild type subunits. For the first time, we develop here models to describe this form of allostery, evaluate them in a simulation study, and test them on experimental data. We show that it is important to consider the assembly reactions in the kinetic model, and to define a formal inhibition scheme. We simulate three inhibition scenarios explicitly, and demonstrate that they result in differing outcomes. Finally, we deduce fitting formulas, and test them on real and simulated data. A non-competitive inhibition formula fitted experimental and simulated data best. To our knowledge, our study is the first one that derives and tests formal allosteric schemes to explain the inhibitory effects of mutant subunits on oligomeric enzymes.     

Effect of Feed Restriction on Performance and Postprandial Nutrient Metabolism in Pigs Co-Infected with Mycoplasma hyopneumoniae and Swine Influenza Virus

As nutritional status and inflammation are strongly connected, feeding and nutritional strategies could be effective to improve the ability of pigs to cope with disease. The aims of this study were to investigate the impact of a feed restriction on the ability of pigs to resist and be tolerant to a coinfection with Mycoplasma hyopneumoniae (Mhp) and the European H1N1 swine influenza virus, and the consequences for nutrient metabolism, with a focus on amino acids. Two groups of specific pathogen-free pigs were inoculated with Mhp and H1N1 21 days apart. One group was fed ad libitum, the other group was subjected to a two-week 40% feed restriction starting one week before H1N1 infection. The two respective mock control groups were included. Three days post-H1N1 infection, 200 g of feed was given to pigs previously fasted overnight and serial blood samples were taken over 4 hours to measure plasma nutrient concentrations. Throughout the study, clinical signs were observed and pathogens were detected in nasal swabs and lung tissues. Feed-restricted pigs presented shorter hyperthermia and a positive mean weight gain over the 3 days post-H1N1 infection whereas animals fed ad libitum lost weight. Both infection and feed restriction reduced postprandial glucose concentrations, indicating changes in glucose metabolism. Post-prandial plasma concentrations of the essential amino acids histidine, arginine and threonine were lower in co-infected pigs suggesting a greater use of those amino acids for metabolic purposes associated with the immune response. Altogether, these results indicate that modifying feeding practices could help to prepare animals to overcome an influenza infection. Connections with metabolism changes are discussed.     

Attachment of blastocysts to lens capsule: A model system for trophoblast-epithelial cell interaction on a natural basement membrane

Summary The bovine lens capsule has previously been shown to provide an optimal surface for the examination of epithelial cell interaction with a basement membrane. This native substrate has been used to investigate some initial aspects of attachment of mouse blastocysts and trophoblastic cellular outgrowth. Mouse blastocysts were presented to the cell-free humoral side of the anterior lens capsule, incubated for 72 h, and examined by scanning and transmission electron microscopy. Blastocysts hatch and attach from their zonae pellucidae by 30 h. Trophoblastic cells proliferate rapidly in a coronal direction, display extensive surface microvilli, and advance by the extension of numerous filipodia, many of which terminate with bulbous projections. These projections were shown by transmission electron microscopy to contain numerous vacuoles and polysomes. To simulate further the initial blastocyst-uterine interaction, a suspension of lens epithelial cells was introduced to the capsule and permitted to form a monolayer prior to the addition of the blastocysts. At 72 h the monolayer of lens cells remained intact. We observed that: a) lens cells appear to recede from the advancing trophoblastic cells, and b) trophoblastic cells extend beneath the monolayer of lens cells and thereby dislodge the cells from the lens capsule substrate. No infiltration of the capsule by the advancing trophoblastic cells was observed. The lens capsule appears to offer a promising system for the study of trophoblast-epithelial cell interaction on a natural basement membrane.     

Modulation of slow and fast elbow extensor EMG tonic activity by stretch reflexes in man

Reflex EMG responses to sudden passive flexion of the elbow were recorded from anconeus and triceps brachii in 5 human volunteers. While the subjects were required not to resist the flexion movement, they were required to maintain an extension torque of 3.5 or 7.0 Nm prior to its onset. Under these isotonic conditions, the latency and amplitude of the reflex activities from anconeus and triceps brachii did not differ significantly, in contrast to the findings of Le Bozec (1986) in actively relaxed subjects. The myotatic/postmyotatic EMG amplitude ratio did not provide a further quantitative way to distinguish between these muscles. The absence of a difference between the reflex activities of a slow (anconeus) and a fast (triceps brachii) muscle is interpreted as resulting from a strong drive of spindle activity on the whole extensor motoneuron pool, which outweights the differences in recruitment due to the differing relative amounts of type I and type II fibres in the two muscles. Differences like those described between finger and calf muscles by other authors are thought to be due to the relative degree of corticalization of these muscles. All short and long latency responses of the muscles increased in magnitude and decreased in latency with increasing background EMG activity as well as with increasing initial length. The position and tonic activity dependency of these responses is explained in terms of alpha-gamma coactivation.     

About the specificity of photoinduced affinity labeling of Escherichia coli ribosomes by dihydrorosaramicin, a macrolide related to erythromycin

Photoactivation of the [3H]dihydrorosaramicin chromophore at a wavelength above 300 nm allows the covalent attachment of the macrolide antibiotic to the bacterial ribosome. Bidimensional electrophoresis shows that the radioactivity is mainly associated with proteins L1, L5, L6, L15, L18, L19, S1, S3, S4, S5 and S9. When photoincorporation of the drug is conducted in the presence of puromycin as effector of [3H]dihydrorosaramicin-binding sites, a decrease in the labeling of most proteins is observed, except for L18 and L19, which are radiolabeled to a larger extent. These results allow us to speculate that L18 and L19 belong to the high-affinity binding site of rosaramicin antibiotic.