Inactivation of Staphylococcus hyicus lipase by hexadecylsulfonyl fluoride: evidence for an active site serine

The Staphylococcus hyicus lipase is an acyl hydrolase with broadsubstrate specificity including neutral glycerides and phospholipids.To obtain further insight into the mechanism of action of thisenzyme, we tested several sulfonyl fluorides as active site-directedinhibitors. The enzyme is resistant to the well-known serineprotease/esterase inhibitor phenylmethanesulfonyl fluoride (PMSF),but is rapidly inactivated by hexadecylsulfonyl fluoride. Thekinetics of inactivation were studied in Triton X-100 micelles.Inactivation is fast and the rate of inactivation is constantover the pH range where this lipase is active. Metal ions likeCa²⁺ and Sr²⁺ do not appreciably influence the rate of inactivation,although the enzymatic activity is significantly increased,suggesting a structural role for these ions. The S.hyicus lipasecontains a consensus sequence G-H/Y-S-X-G. Substitution by site-directedmutagenesis of this serine (Ser369) by a cysteine resulted ina mutant with only 0.2% residual activity. The activity of thismutant could not be inhibited with water-soluble sulfhydrylreagents either in the presence or absence of Triton X-100 micelles.In the presence of Triton X-100 micelles, inactivation of themutant occurred with 4-nitrophenylhexadecyl disulfide (t_1/2= 125 min) while the wild-type enzyme does not react at all.We conclude that Ser369 is the active site residue and thatin water this residue is inaccessible. Only after interfacialactivation Ser369 (or Cys369) becomes exposed and reacts withirreversible inhibitors.     

Contribution of the C-terminal amino acid to the stability of Bacillus subtilis neutral protease

The role of the C-terminal Leu300 in maintaining thermal stabilityof the neutral protease of Bacillus subtilis was investigated.From model building studies based on the three dimensional structureof thermolysin, the neutral protease of B.thermoproteolyticus,it was conduded that this residue is located in a hydrophobicpocket composed of residues located in the C-terminal and themiddle domain. To test the hypothesis that Leu300, by contributingto a stabilizing interaction between these domains, is importantfor enzyme stability, several neutral protease mutants wereconstructed and characterized. The thermostability of the enzymewas lowered by deleting Leu300 or by replacing this residueby a smaller (Ala), a polar (Asn) or a sterically unfavourable(He) amino acid. Thermostabiity was increased upon replacingLeu300 by Phe. These results are in agreement with model-buildingstudies. The effects on thermostability observed after mutatingthe corresponding Val318 in the thermostable neutral proteaseof B.stearothermophilus were less pronounced.     

Subsoil <Superscript>15</Superscript>N-N<Subscript>2</Subscript>O Concentrations in a Sandy Soil Profile After Application of <Superscript>15</Superscript>N-fertilizer

Studies on emissions of nitrous oxide (N₂O) from agricultural soils mostly focus on fluxes between the soil and the atmosphere or are limited to the atmosphere in the topsoil. However, in soils with shallow water tables, significant N₂O formation may occur closer to the groundwater. The aims of this study were (i) to determine the importance of subsoil N₂O formation in a sandy soil; and (ii) to obtain a quantitative insight in the contribution of subsoil N₂O to the overall losses of N₂O to the environment. We applied ¹⁵N labeled fertilizer at a rate of 5.22 kg ¹⁵N ha⁻¹; 50% as Ca(NO₃)₂ and 50% as NH₄Cl, on a mesic typic Haplaquod seeded with potatoes (Solanum tuberosum L.), and traced soil N₂O concentrations and fluxes over a one-year period. Throughout the year, total N₂O and the amount of ¹⁵N recovered in soil N₂O were highest in the subsoil, with a maximum concentration at 48 cm depth in mid-February of 19900 μl m⁻³ and 24 μg ¹⁵N m⁻³, respectively. The maximum concentration coincided with the highest water-filled pore space of 71%. The cumulative flux of N₂O was 446 g N₂O-N ha⁻¹, the recovery of ¹⁵N in this flux was 0.06%. During the summer, maximum fluxes followed high soil N₂O concentrations. During winter, no such relation was found. We concluded that the formation of N₂O was the highest in the subsoil, largely controlled by water-filled pore space rather than NO₃⁻ concentration or temperature. Although high subsoil N₂O concentrations did not lead to high surface fluxes of N₂O in the winter, artificial draining may lead to high indirect N₂O emissions through supersaturated drainage water.     

Rheological behaviour of multiphase slurries for combustion applications

The rheological behaviour of a concentrated coal-water-fuel oil slurry was investigated with a tube viscometer, to optimize its formulation. The non-Newtonian character of the slurry follows the rheological model of Ostwald de Waele, according to the experimental results obtained. A factorial plan was used and empirical equations were obtained which correlate rheological characteristics with the mass fractions of the slurry components.     

Influence of Tempering Temperature on Cyclic Viscoplastic Behaviour of 55NiCrMoV7 Steel

Low cycle fatigue behaviour of a steel 55NiCrMoV7 under tour tempered conditions is reported. One special type of total swain controlled isothermal cyclic deformation tests were performed in the temperature range 20℃ to 600℃ for the steel tempered 2h at 350℃, 460℃, 560℃ and 600℃. The influence of temperature on cyclic behaviour was investigated. Generally, the cyclic stress response shows an initial exponential softening for the first few cycles, followed by a gradual softening without cyclic softening saturatioo. At 10^-2 strain rate, σmax.Δσ／2 decrease with the test tumperature for all hardness levels. They decrease linearly with tempering temperature when testing temperature is lower than that of tempering, but rest nearly constant when test temperature is equal to or exceed tempering temperature of steel. Cyclic softening intensity increases with testing temperature from 300℃ to 600℃, but the maximal softening intensity occurs at room tumpemture. The strain rate influences notably the cyclic behavioor when T≥50℃, The time dependence of cyclic behaviour is closely related to test temperature and the tempering history of the steel.     

Performance control for interconnection of identical systems: Application to PLL network design

In this paper, the problem of the control law design for interconnected identical systems ensuring the global stability and the global performance properties is under consideration. Inspired by the decentralized control law design methodology using the dissipativity input–output approach, the problem is reduced to the problem of satisfying two conditions: (i) the condition on the interconnection and (ii) the condition on the local subsystem dynamics. Both problems are efficiently solved applying a (quasi‐) convex LMI optimization and standard H_∞ synthesis. The proposed design methodology is applied to the control law design of a synchronous PLL network. Copyright © 2014 John Wiley & Sons, Ltd.     

Proteomics of plaques and novel sources of potential biomarkers for atherosclerosis

Cardiovascular disease (CVD) is the leading cause of death and loss of productive life years in the world. The underlying syndrome of CVD, atherosclerosis, is a complex disease process, which involves lipid metabolism, inflammation, innate and adaptive immunity, and many other pathophysiological aspects. Furthermore, CVD is influenced by genetic as well as environmental factors. Early detection of CVD and identification of patients at risk are crucial to reduce the burden of disease and to allow personalized treatment. As established risk factors fail to accurately predict which part of the population is likely to suffer from the disease, novel biomarkers are urgently needed. Proteomics can play a significant role in identifying these biomarkers. In this review, we describe the progress made in proteome profiling of the atherosclerotic plaque and several novel sources of potential biomarkers, including circulating cells and plasma extracellular vesicles. The importance of longitudinal biobanking in biomarker discovery is highlighted and exemplified by several plaque proteins identified in the biobank study Athero-Express. Finally, we discuss the PTMs of proteins that are involved in atherosclerosis, which may become one of the foci in the ongoing quest for biomarkers through proteomics of plaque and other matrices relevant to the progression of atherosclerosis.     

The design of a collision avoidance system for use by Pilots operating on the airport ramp and in taxiway areas

Ground collisions have serious implications from both a safety and a commercial perspective. This paper reports on human computer interaction (HCI) research related to the advancement of a collision avoidance system, for use by Pilots operating on the airport ramp and in taxiway areas. Primarily, this paper focuses on the key findings of this research and the emerging HCI design solution.     

Interaction of transport resistances with biochemical reaction in packed-bed solid-state fermentors: effect of temperature gradients

Subdermal superficial liposuction, first presented by the authors at the ISAPS Congress at Zurich in 1989, is performed with thin three-hole Mercedes cannulas (diameter ranges from 1.8 to 2 mm) to treat small and secondary adiposities and to allow better skin retraction. Suction of the subdermal layer of fat reduces the thickness and consistency of the superficial fat and enhances the possibility of skin retraction. In cases where there is a large adiposity of the abdomen, arms, or inner thighs, there is a conspicuous volume of fat whose weight tends to overstretch and to carry the overlying skin downward. In these cases we need to reduce the large fat volume to permit effective skin retraction. Therefore, we apply the principles of traditional liposuction with those of subdermal superficial liposuction to aspirate large amounts of fat from all the adipose layers. We call this technique Massive All Layer Liposuction (MALL). The amount of skin shrinkage after this "defatting" procedure is remarkable and the clinical results are very good. The MALL technique can be applied to other areas as well. In our experience this new liposuction technique has dramatically reduced the indications of abdominoplasties and dermolipectomies of inner thighs and arms.     

Thin and thick YBCO films grown by MOCVD processes. Study of the angular magnetic field dependence of current transport properties

Thin and thick YBCO Films have been grown by Aerosol Assisted as well as by thermal MOCVD. The Aerosol Assisted MOCVD technique allows the growth of YBCO films from a single liquid source at deposition rates of up to 10m/h.Transport measurements (I-V) on etched microbridges using a single pulse technique have been performed. The angular magnetic field dependence J_c() of the critical current density from T_c down to 50 K has been measured. Transport properties are reported and discussed with respect to the microstructural features as determined by X-ray diffraction and electron microscopy.