Methionine sulfoxide reductases are essential for virulence of Salmonella typhimurium

Production of reactive oxygen species represents a fundamental innate defense against microbes in a diversity of host organisms. Oxidative stress, amongst others, converts peptidyl and free methionine to a mixture of methionine-S- (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO). To cope with such oxidative damage, methionine sulfoxide reductases MsrA and MsrB are known to reduce MetSOs, the former being specific for the S-form and the latter being specific for the R-form. However, at present the role of methionine sulfoxide reductases in the pathogenesis of intracellular bacterial pathogens has not been fully detailed. Here we show that deletion of msrA in the facultative intracellular pathogen Salmonella (S.) enterica serovar Typhimurium increased susceptibility to exogenous H(2)O(2), and reduced bacterial replication inside activated macrophages, and in mice. In contrast, a ΔmsrB mutant showed the wild type phenotype. Recombinant MsrA was active against free and peptidyl Met-S-SO, whereas recombinant MsrB was only weakly active and specific for peptidyl Met-R-SO. This raised the question of whether an additional Met-R-SO reductase could play a role in the oxidative stress response of S. Typhimurium. MsrC is a methionine sulfoxide reductase previously shown to be specific for free Met-R-SO in Escherichia (E.) coli. We tested a ΔmsrC single mutant and a ΔmsrBΔmsrC double mutant under various stress conditions, and found that MsrC is essential for survival of S. Typhimurium following exposure to H(2)O(2,) as well as for growth in macrophages, and in mice. Hence, this study demonstrates that all three methionine sulfoxide reductases, MsrA, MsrB and MsrC, facilitate growth of a canonical intracellular pathogen during infection. Interestingly MsrC is specific for the repair of free methionine sulfoxide, pointing to an important role of this pathway in the oxidative stress response of Salmonella Typhimurium.     

Pancreatic Duct Obstruction in the Pig: Light Microscopy of Chronic Pancreatitis

Morphological changes of pancreatic tissue in young pigs caused by surgical ligation of the main pancreatic duct are described. Nineteen animals from 6 to 7 weeks in age were operated on and necropsied 3 or 6 to 8 weeks later. Twelve pigs developed a pronounced chronic pancreatitis with complete exocrine insufficiency. Of the 7 animals failing to develop ectasia of pancreatic ducts, 2 died due to surgical complications. In addition, 3 pigs were sham-operated and served as controls. In macroscopical studies it was observed that in the pronounced pancreatitis cases the ligated duct was greatly dilated by a clear watery fluid. Only remnants of pale and firm grandular tissues were seen around the ectatic ducts. Microscopically, typical changes of chronic pancreatitis were noted. Complete disappearance of acini was followed by ductular cell proliferations. Glandular tissues were divided into lobuli by fibrotic tissues and fat cells. The wall of the main pancreatic duct was greatly thickened and fibrotic, presenting intensely proliferating ductular cells and round cell infiltrates. Furthermore, enlarged endocrine islets surrounded by connective tissue fibres were seen.     

Direct Measurement of the Cortical Tension during the Growth of Membrane Blebs

Mechanics is at the heart of many cellular processes and its importance has received considerable attention during the last two decades. In particular, the tension of cell membranes, and more specifically of the cell cortex, is a key parameter that determines the mechanical behavior of the cell periphery. However, the measurement of tension remains challenging due to its dynamic nature. Here we show that a noninvasive interferometric technique can reveal time-resolved effective tension measurements by a high-accuracy determination of edge fluctuations in expanding cell blebs of filamin-deficient melanoma cells. The introduced technique shows that the bleb tension is ∼10–100 pN/μm and increases during bleb growth. Our results directly confirm that the subsequent stop of bleb growth is induced by an increase of measured tension, possibly mediated by the repolymerized actin cytoskeleton.     

Strain while skiing and hauling a sledge or carrying a backpack

Eight soldiers on skis transported three loads of different weights on the level, uphill and downhill. The load was placed either on a cargo sledge or in a backpack or divided between the sledge and the backpack. The sledge had a new type of haulage-shaft, which was fixed to both sides of the pelvis. A service belt spread the pull over the whole upper body. The physical stain of different transport methods and the serviceability of the sledge was studied by measuring heart rate (HR), oxygen consumption, ventilation, and perceived exertion. The results indicate that both absolute and relative strain were systematically lower when pulling the load on the sledge than when carrying it in the backpack and on the sledge. HR when pulling a load equal to the human body weight on the sledge was on average 133 beats X min-1; HR was significantly higher 144 beats X min-1 when the load was divided between backpack and sledge. At the lower load level the differences between the transport methods were not significant for HR, oxygen consumption or ventilation. Uphill travel increased oxygen consumption by about 50% over that on the level. Perceived exertion at all load levels was significantly lower with the sledge than with the backpack alone or in combination. The estimated maximal allowable working time emphasized the advantage of the sledge and the importance of high physical working capacity. The maneuverability of the sledge with the new haulage shaft was good and the braking mechanisms worked well.(ABSTRACT TRUNCATED AT 250 WORDS)     

Matrix Metalloproteinases -8 and -9 and Tissue Inhibitor of Metalloproteinase-1 in Burn Patients. A Prospective Observational Study

IntroductionMatrix metalloproteinases (MMPs) -8 and -9 are released from neutrophils in acute inflammation and may contribute to permeability changes in burn injury. In retrospective studies on sepsis, levels of MMP-8, MMP-9, and tissue inhibitor of metalloproteinase-1 (TIMP-1) differed from those of healthy controls, and TIMP-1 showed an association with outcome. Our objective was to investigate the relationship between these proteins and disease severity and outcome in burn patients.MethodsIn this prospective, observational, two-center study, we collected plasma samples from admission to day 21 post-burn, and burn blister fluid samples on admission. We compared MMP-8, -9, and TIMP-1 levels between TBSA<20% (N = 19) and TBSA>20% (N = 30) injured patients and healthy controls, and between 90-day survivors and non-survivors. MMP-8, -9, and TIMP-1 levels at 24-48 hours from injury, their maximal levels, and their time-adjusted means were compared between groups. Correlations with clinical parameters and the extent of burn were analyzed. MMP-8, -9, and TIMP-1 levels in burn blister fluids were also studied.ResultsPlasma MMP-8 and -9 were higher in patients than in healthy controls (P<0.001 and P = 0.016), but only MMP-8 differed between the TBSA<20% and TBSA>20% groups. MMP-8 and -9 were not associated with clinical severity or outcome measures. TIMP-1 differed significantly between patients and controls (P<0.001) and between TBSA<20% and TBSA>20% groups (P<0.002). TIMP-1 was associated with 90-day mortality and correlated with the extent of injury and clinical measures of disease severity. TIMP-1 may serve as a new biomarker in outcome prognostication of burn patients.     

Optical study of DNA surface hybridization reveals DNA surface density as a key parameter for microarray hybridization kinetics

We investigate the kinetics of DNA hybridization reactions on glass substrates, where one 22 mer strand (bound-DNA) is immobilized via phenylene-diisothiocyanate linker molecule on the substrate, the dye-labeled (Cy3) complementary strand (free-DNA) is in solution in a reaction chamber. We use total internal reflection fluorescence for surface detection of hybridization. As a new feature we perform a simultaneous real-time measurement of the change of free-DNA concentration in bulk parallel to the total internal reflection fluorescence measurement. We observe that the free-DNA concentration decreases considerably during hybridization. We show how the standard Langmuir kinetics needs to be extended to take into account the change in bulk concentration and explain our experimental results. Connecting both measurements we can estimate the surface density of accessible, immobilized bound-DNA. We discuss the implications with respect to DNA microarray detection.     

Poly(ADP-Ribose) polymerase inhibition improves endothelial dysfunction induced by hypochlorite

Reactive oxygen species, such as myeloperoxidase-derived hypochlorite, induce oxidative stress and DNA injury. The subsequent activation of the DNA-damage-poly(ADP-ribose) polymerase (PARP) pathway has been implicated in the pathogenesis of various diseases, including ischemia-reperfusion injury, circulatory shock, diabetic complications, and atherosclerosis. We investigated the effect of PARP inhibition on the impaired endothelium-dependent vasorelaxation induced by hypochlorite. In organ bath experiments for isometric tension, we investigated the endothelium-dependent and endothelium-independent vasorelaxation of isolated rat aortic rings using cumulative concentrations of acetylcholine and sodium nitro-prusside. Endothelial dysfunction was induced by exposing rings to hypochlorite (100-400 microM). In the treatment group, rings were preincubated with the PARP inhibitor INO-1001. DNA strand breaks were assessed by the TUNEL method. Immunohistochemistry was performed for 4-hydroxynonenal (a marker of lipid peroxidation), nitrotyrosine (a marker of nitrosative stress), and poly(ADP-ribose) (an enzymatic product of PARP). Exposure to hypochlorite resulted in a dose-dependent impairment of endothelium-dependent vasorelaxation of aortic rings, which was significantly improved by PARP inhibition, whereas the endothelium-independent vasorelaxation remained unaffected. In the hypochlorite groups we found increased DNA breakage, lipidperoxidation, and enhanced nitrotyrosine formation. The hypochloride-induced activation of PARP was prevented by INO-1001. Our results demonstrate that PARP activation contributes to the pathogenesis of hypochlorite-induced endothelial dysfunction, which can be prevented by PARP inhibitors.     

Ecosystem services–A tool for sustainable management of human–environment systems. Case study Finnish Forest Lapland

The concept of ecosystem services (ESs) is a relatively new scientific methodology, offering a possible approach to the prevention of ecological problems caused by human action and to the resolution of conflicts arising from land-use questions. Since ESs were launched as a major conceptual tool in the Millennium Ecosystem Assessment (MA, 2005), interest in them has been increasing. Despite the scientific as well as economic and political enthusiasm for the ES approach, only few case studies have as yet been published. We studied the interface between ESs and landscape planning in Forest Lapland, in northern Finland. In the article, we present a methodology and various databases which can be used in applied research on ESs. We classify the ESs offered by various biotopes of the study area, and examine the effects of different land-use forms on the provision of ESs. On the basis of our results, we suggest possible uses of the European CORINE land cover database in case studies.     

Response of zooplankton to nutrient enrichment and fish in shallow lakes: a pan-European mesocosm experiment

1. Responses of zooplankton to nutrient enrichment and fish predation were studied in 1998 and 1999 by carrying out parallel mesocosm experiments in six lakes across Europe. 2. Zooplankton community structure, biomass and responses to nutrient and fish manipulation showed geographical and year‐to‐year differences. Fish had a greater influence than nutrients in regulating zooplankton biomass and especially the relative abundances of different functional groups of zooplankton. When fish reduced the biomass of large crustaceans, there was a complementary increase in the biomasses of smaller crustacean species and rotifers. 3. High abundance of submerged macrophytes provided refuge for zooplankton against fish predation but this refuge effect differed notably in magnitude among sites. 4. Large crustacean grazers (Daphnia, Diaphanosoma, Sida and Simocephalus) were crucial in controlling algal biomass, while smaller crustacean grazers and rotifers were of minor importance. Large grazers were able to control phytoplankton biomass even under hypereutrophic conditions (up to 1600 μg TP L^?1) when grazer biomass was high (>80–90 μg dry mass L^?1) or accounted for >30% of the grazer community. 5. The littoral zooplankton community was less resistant to change following nutrient enrichment in southern Spain, at high temperatures (close to 30 °C), than at lower temperatures (17–23 °C) characterising the other sites. This lower resistance was because of a greater importance of nutrients than zooplankton in controlling algal biomass. 6. Apart from the reduced role of large crustacean grazers at the lowest latitude, no consistent geographical patterns were observed in the responses of zooplankton communities to nutrient and fish manipulation.