Mechanism of the Escherichia coli DNA T:G-mismatch endonuclease (Vsr protein) probed with thiophosphate-containing oligodeoxynucleotides

The mechanism of the Escherichia coli DNA T:G mismatch endonuclease (Vsr) has been investigated using oligodeoxynucleotides substituted, at the scissile phosphate, with isomeric phosphorothioates and a 3'-phosphorothiolate. Binding and kinetic data with the phosphorothioates/phosphorothiolate indicate that the two magnesium ions, which constitute essential co-factors, are required to stabilise the extra negative charge developed on the phosphate as the transition state is formed. Additionally one of the magnesium ions serves to activate the leaving group (the non-bridging 3'-oxygen atom of the scissile phosphate) during the hydrolysis reaction. Stereochemical analysis, using the R-p phosphorothioate isomer, indicates that Vsr carries out a hydrolytic reaction with inversion of stereochemistry at phosphorus, compatible with an in-line attack of water and a pentacovalent transition state with trigonal bipyramidal geometry. In conjunction with structures of Vsr bound to its products, these data allow the reconstruction of the enzyme-substrate complex and a comprehensive description of the hydrolysis mechanism. (c) 2005 Elsevier Ltd. All rights reserved.

Is a native rodent competitively dominant over an invasive rodent in lowland agro-forest habitat of the Philippines?

In the lowland agro-forest of the Sierra Madre Biodiversity Corridor (SMBC), it is considered that a native rodent species, Rattus everetti is competitively dominant over an invasive pest species, Rattus tanezumi. The main aim of this study was to assess the response of R. tanezumi following short term removal of R. everetti. We tested this experimentally by trapping and removing R. everetti from two treatment sites in agro-forest habitat on three occasions over three consecutive months. This was followed by three months of non-removal trapping. Two non-treatment sites were trapped for comparison. Following R. everetti removal, R. everetti individuals rapidly immigrated into the treatment sites and a significantly higher proportion of R. tanezumi females were in breeding condition in the treatment sites than in the non-treatment sites. The results from this study provide evidence of competition between native and invasive rodent species in complex agro-ecosystems. We were also able to demonstrate that R. everetti populations are able to recover rapidly from the non-target effects of short-term lethal control in and around agro-forest.

Age related changes in speed and mechanism of adult skeletal muscle stem cell migration

Skeletal muscle undergoes a progressive age-related loss in mass and function. Preservation of muscle mass depends in part on satellite cells, the resident stem cells of skeletal muscle. Reduced satellite cell function may contribute to the age-associated decrease in muscle mass. Here we focused on characterising the effect of age on satellite cell migration. We report that aged satellite cells migrate at less than half the speed of young cells. In addition, aged cells show abnormal membrane extension and retraction characteristics required for amoeboid based cell migration. Aged satellite cells displayed low levels of integrin expression. By deploying a mathematical model approach to investigate mechanism of migration, we have found that young satellite cells move in a random ‘memoryless’ manner whereas old cells demonstrate superdiffusive tendencies. Most importantly, we show that nitric oxide, a key regulator of cell migration, reversed the loss in migration speed and reinstated the unbiased mechanism of movement in aged satellite cells. Finally we found that although Hepatocyte Growth Factor increased the rate of aged satellite cell movement it did not restore the memoryless migration characteristics displayed in young cells. Our study shows that satellite cell migration, a key component of skeletal muscle regeneration, is compromised during aging. However, we propose clinically approved drugs could be used to overcome these detrimental changes.

Assessing the microbial oxidative stress mechanism of ozone treatment through the responses of Escherichia coli mutants

Aims: To investigate the effect of the oxidative stress of ozone on the microbial inactivation, cell membrane integrity and permeability and morphology changes of Escherichia coli. Methods and Results: Escherichia coli BW 25113 and its isogenic mutants in soxR, soxS, oxyR, rpoS and dnaK genes were treated with ozone at a concentration of 6 lg ml)1 for a period up to 240 s. A significant effect of ozone exposure on microbial inactivation was observed. After ozonation, minor effects on the cell membrane integrity and permeability were observed, while scanning electron microscopy analysis showed slightly altered cell surface structure. Conclusions: The results of this study suggest that cell lysis was not the major mechanism of microbial inactivation. The deletion of oxidative stress–related genes resulted in increased susceptibility of E. coli cells to ozone treatment, implying that they play an important role for protection against the radicals produced by ozone. However, DnaK that has previously been shown to protect against oxidative stress did not protect against ozone treatment in this study. Furthermore, RpoS was important for the survival against ozone. Significance and Impact of the Study: This study provides important information about the role of oxidative stress in the responses of E. coli during ozonation.

Cyclic extrusion of a lava dome based on a stick-slip mechanism

Lava dome eruptions are sometimes characterised by large periodic fluctuations in extrusion rate over periods of hours that may be accompanied by Vulcanian explosions and pyroclastic flows. We consider a simple system of nonlinear equations describing a 1D flow of lava extrusion through a deep elastic dyke feeding a shallower cylindrical conduit in order to simulate this short-period cyclicity. Stick-slip conditions depending on a critical shear stress are assumed at the wall boundary of the cylindrical conduit. By analogy with the behaviour of industrial polymers in a plastic extruder, the elastic dyke acts like a barrel and the shallower cylindrical portion of the conduit as a die for the flow of magma acting as a polymer. When we applied the model to the Soufrière Hills Volcano, Montserrat, for which the key parameters have been evaluated from previous studies, cyclic extrusions with periods from 3 to 30 h were readily simulated, matching observations. The model also reproduces the reduced period of cycles observed when a major unloading event occurs due to lava dome collapse.

The influence of Langmuir turbulence on the scaling for the dissipation rate in the oceanic boundary layer

A model for estimating the turbulent kinetic energy dissipation rate in the oceanic boundary layer, based on insights from rapid-distortion theory, is presented and tested. This model provides a possible explanation for the very high dissipation levels found by numerous authors near the surface. It is conceived that turbulence, injected into the water by breaking waves, is subsequently amplified due to its distortion by the mean shear of the wind-induced current and straining by the Stokes drift of surface waves. The partition of the turbulent shear stress into a shear-induced part and a wave-induced part is taken into account. In this picture, dissipation enhancement results from the same mechanism responsible for Langmuir circulations. Apart from a dimensionless depth and an eddy turn-over time, the dimensionless dissipation rate depends on the wave slope and wave age, which may be encapsulated in the turbulent Langmuir number La_t. For large La_t, or any Lat but large depth, the dissipation rate tends to the usual surface layer scaling, whereas when Lat is small, it is strongly enhanced near the surface, growing asymptotically as ɛ ∝ La_t^{-2} when La_t → 0. Results from this model are compared with observations from the WAVES and SWADE data sets, assuming that this is the dominant dissipation mechanism acting in the ocean surface layer and statistical measures of the corresponding fit indicate a substantial improvement over previous theoretical models. Comparisons are also carried out against more recent measurements, showing good order-of-magnitude agreement, even when shallow-water effects are important.

Allosteric mechanism controls traffic in the chaperone/usher pathway

Many virulence organelles of Gram-negative bacterial pathogens are assembled via the chaperone/ usher pathway. The chaperone transports organelle subunits across the periplasm to the outer membrane usher, where they are released and incorporated into growing fibers. Here, we elucidate the mechanism of the usher-targeting step in assembly of the Yersinia pestis F1 capsule at the atomic level. The usher interacts almost exclusively with the chaperone in the chaperone:subunit complex. In free chaperone, a pair of conserved proline residues at the beginning of the subunit-binding loop form a ‘‘proline lock’’ that occludes the usher-binding surface and blocks usher binding. Binding of the subunit to the chaperone rotates the proline lock away from the usher-binding surface, allowing the chaperone-subunit complex to bind to the usher. We show that the proline lock exists in other chaperone/usher systems and represents a general allosteric mechanism for selective targeting of chaperone:subunit complexes to the usher and for release and recycling of the free chaperone.

Pathogenic LRRK2 mutations do not alter gene expression in cell model systems or human brain tissue

Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances.

c-Cbl mediates ubiquitination, degradation, and down-regulation of human protease-activated receptor 2

Mechanisms that arrest G-protein-coupled receptor (GPCR) signaling prevent uncontrolled stimulation that could cause disease. Although uncoupling from heterotrimeric G-proteins, which transiently arrests signaling, is well described, little is known about the mechanisms that permanently arrest signaling. Here we reported on the mechanisms that terminate signaling by protease-activated receptor 2 (PAR(2)), which mediated the proinflammatory and nociceptive actions of proteases. Given its irreversible mechanism of proteolytic activation, PAR(2) is a model to study the permanent arrest of GPCR signaling. By immunoprecipitation and immunoblotting, we observed that activated PAR(2) was mono-ubiquitinated. Immunofluorescence indicated that activated PAR(2) translocated from the plasma membrane to early endosomes and lysosomes where it was degraded, as determined by immunoblotting. Mutant PAR(2) lacking intracellular lysine residues (PAR(2)Delta14K/R) was expressed at the plasma membrane and signaled normally but was not ubiquitinated. Activated PAR(2) Delta14K/R internalized but was retained in early endosomes and avoided lysosomal degradation. Activation of wild type PAR(2) stimulated tyrosine phosphorylation of the ubiquitin-protein isopeptide ligase c-Cbl and promoted its interaction with PAR(2) at the plasma membrane and in endosomes in an Src-dependent manner. Dominant negative c-Cbl lacking the ring finger domain inhibited PAR(2) ubiquitination and induced retention in early endosomes, thereby impeding lysosomal degradation. Although wild type PAR(2) was degraded, and recovery of agonist responses required synthesis of new receptors, lysine mutation and dominant negative c-Cbl impeded receptor ubiquitination and degradation and allowed PAR(2) to recycle and continue to signal. Thus, c-Cbl mediated ubiquitination and lysosomal degradation of PAR(2) to irrevocably terminate signaling by this and perhaps other GPCRs.

Suppression of a pro-apoptotic K+ channel as a mechanism for hepatitis C virus persistence

An estimated 3% of the global population are infected with hepatitis C virus (HCV), and the majority of these individuals will develop chronic liver disease. As with other chronic viruses, establishment of persistent infection requires that HCV-infected cells must be refractory to a range of pro-apoptotic stimuli. In response to oxidative stress, amplification of an outward K(+) current mediated by the Kv2.1 channel, precedes the onset of apoptosis. We show here that in human hepatoma cells either infected with HCV or harboring an HCV subgenomic replicon, oxidative stress failed to initiate apoptosis via Kv2.1. The HCV NS5A protein mediated this effect by inhibiting oxidative stress-induced p38 MAPK phosphorylation of Kv2.1. The inhibition of a host cell K(+) channel by a viral protein is a hitherto undescribed viral anti-apoptotic mechanism and represents a potential target for antiviral therapy.

A mechanism for Atlantic multidecadal variability in the Kiel Climate Model

Atlantic Multidecadal Variability (AMV) is investigated in a millennial control simulation with the Kiel Climate Model (KCM), a coupled atmosphere–ocean–sea ice model. An oscillatory mode with approximately 60 years period and characteristics similar to observations is identified with the aid of three-dimensional temperature and salinity joint empirical orthogonal function analysis. The mode explains 30 % of variability on centennial and shorter timescales in the upper 2,000 m of the North Atlantic. It is associated with changes in the Atlantic Meridional Overturning Circulation (AMOC) of ±1–2 Sv and Atlantic Sea Surface Temperature (SST) of ±0.2 °C. AMV in KCM results from an out-of-phase interaction between horizontal and vertical ocean circulation, coupled through Irminger Sea convection. Wintertime convection in this region is mainly controlled by salinity anomalies transported by the Subpolar Gyre (SPG). Increased (decreased) dense water formation in this region leads to a stronger (weaker) AMOC after 15 years, and this in turn leads to a weaker (stronger) SPG after another 15 years. The key role of salinity variations in the subpolar North Atlantic for AMV is confirmed in a 1,000 year long simulation with salinity restored to model climatology: No low frequency variations in convection are simulated, and the 60 year mode of variability is absent.

Proposal of redundant drive wire mechanism for producing motions with high acceleration and high precision

A new wire mechanism called Redundant Drive Wire Mechanism (RDWM) is proposed. The purpose of this paper is to build up the theory of a RDWM with fast motion and fine motion. First, the basic concepts of the proposed mechanism is presented. Second, the vector closure condition for the proposed mechanism is developed. Next, we present the basic equations, propose the basic structure of RDWM with the Internal DOF module, Double Actuation Modules and Precision Modules together with the properties of the mechanism. Finally, we conduct the simulation to show the validity of the RDWM.

A robust mechanism for strengthening of the Brewer–Dobson circulation in response to climate change: critical-layer control of subtropical wave breaking

Climate models consistently predict a strengthened Brewer–Dobson circulation in response to greenhouse gas (GHG)-induced climate change. Although the predicted circulation changes are clearly the result of changes in stratospheric wave drag, the mechanism behind the wave-drag changes remains unclear. Here, simulations from a chemistry–climate model are analyzed to show that the changes in resolved wave drag are largely explainable in terms of a simple and robust dynamical mechanism, namely changes in the location of critical layers within the subtropical lower stratosphere, which are known from observations to control the spatial distribution of Rossby wave breaking. In particular, the strengthening of the upper flanks of the subtropical jets that is robustly expected from GHG-induced tropospheric warming pushes the critical layers (and the associated regions of wave drag) upward, allowing more wave activity to penetrate into the subtropical lower stratosphere. Because the subtropics represent the critical region for wave driving of the Brewer–Dobson circulation, the circulation is thereby strengthened. Transient planetary-scale waves and synoptic-scale waves generated by baroclinic instability are both found to play a crucial role in this process. Changes in stationary planetary wave drag are not so important because they largely occur away from subtropical latitudes.