Characterization of three pyranose dehydrogenase isoforms from the litter-decomposing basidiomycete Leucoagaricus meleagris (syn. Agaricus meleagris)

Nicht verfügbarMultigenicity is commonly found in fungal enzyme systems, with the purpose of functional compensation upon deficiency of one of its members or leading to enzyme isoforms with new functionalities through gene diversification. Three genes of the flavin-dependent glucosemethanolcholine (GMC) oxidoreductase pyranose dehydrogenase (AmPDH) were previously identified in the litter-degrading fungus Agaricus (Leucoagaricus) meleagris, of which only AmPDH1 was successfully expressed and characterized. The aim of this work was to study the biophysical and biochemical properties of AmPDH2 and AmPDH3 and compare them with those of AmPDH1. AmPDH1, AmPDH2 and AmPDH3 showed negligible oxygen reactivity and possess a covalently tethered FAD cofactor. All three isoforms can oxidise a range of different monosaccarides and oligosaccharides including glucose, mannose, galactose and xylose, which are the main constituent sugars of cellulose and hemicelluloses, and judging from the apparent steady-state kinetics determined for these sugars, the three isoforms do not show significant differences pertaining to their reaction with sugar substrates. They oxidize glucose both at C2 and C3 and upon prolonged reaction C2 and C3 double-oxidized glucose is obtained, confirming that the A. meleagris genes pdh2 (AY753308.1) and pdh3 (DQ117577.1) indeed encode CAZy class AA3_2 pyranose dehydrogenases. While reactivity with electron donor substrates was comparable for the three AmPDH isoforms, their kinetic properties differed significantly for the model electron acceptor substrates tested, a radical (the 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] cation radical), a quinone (benzoquinone) and a complexed iron ion (the ferricenium ion). Thus, a possible explanation for this PDH multiplicity in A. meleagris could be that different isoforms react preferentially with structurally different electron acceptors in vivo.(VLID)192910

Time-resolved temperature measurements during pulsed laser irradiation uslng thin film metal thermometers

In this article, we describe a technique using Nisi and Pt thin film metal thermometers to provide accurate temperature information on a nanosecond time scale during pulsed laser processing of materials. A surface layer of interest is deposited onto the thermometer layer, and temperatures are determined from temperature dependent changes in the metal film's resistance. Details concerning the design and fabrication of the device structure and experimental considerations in making nanosecond resolved resistance measurements are discussed. Simple analytical estimates are presented to extract quantities such as incident laser energy stored in the sample. Finally, transient temperature data in the thermometer film, in combination with heat flow calculations, allow temperature determination as a function of time and depth into the sample and, additionally, can provide information about material properties of the surface layer

The role of cell volume regulation and chloride currents during phagocytosis in microglia

eingereicht von Kittl MichaelLiteraturverzeichnis: Blatt 19-24Abstract in deutscher und englischer SpracheParis-Lodron-Universität Salzburg, Dissertation, 2019OeBB(VLID)464456

The Modified Lemaire Procedure

Background: Anterolateral rotatory instability (ALRI) may be one reasons why anterior cruciate ligament (ACL) reconstructions fail. An additional reconstruction of the anterolateral structures reduces the graft rupture rate by 50%. The modified Lemaire procedure is one of the lateral extra-articular tenodeses (LET) to restrain ALRI. The purpose of the present video is to describe this technique in detail. Indications: According to the international anterolateral complex consensus group indications may include revision ACL reconstruction, high grade pivot shift, generalized ligamentous laxity, like genu recurvatum, and young patients returning to pivoting activities. However, clinical evidence to recommend specific indications is still missing. Technique description: A 7 to 10 mm wide strip of the iliotibial tract, attached to Gerdy tubercle is shuttled deep to the lateral collateral ligament and is then attached proximal to the lateral femoral epicondyle. The biomechanical principle behind this is to place the graft posterior to the transverse axis of rotation through the entire range of motion. This posterior pull will restrain internal rotation and the anterior subluxation of the lateral tibial plateau. Results: Newer comparative studies show a reduced graft rupture rate and higher rate of returning to preinjury level, when adding an anterolateral extra-articular reconstruction to the ACL reconstruction at 2 years follow-up. At long-term follow-up there was also a trend toward decreased graft rupture rate. However, one must be aware of the possible increased risk of lateral compartment osteoarthritis. Conclusion: The modified Lemaire procedure is an easy-to-use addon to the ACL reconstruction, which can effectively reduce graft failure rate

Time-Resolved Temperature Measurements during Rapid Solidification of Si-As Alloys Induced by Pulsed-Laser Melting.

The solidification of Si-As alloys induced by pulsed-laser melting was studied at regrowth velocities where the partition coefficient is close to unity. The congruent melting temperatures T0 of Si-As alloys were determined using a temperature measurement technique developed for this work, and was confirmed with T0 measurements using three other methods. The time-resolved temperature measurement uses a thin-film platinum thermistor, below and electrically isolated from the Si-As alloy layer, to directly measure the temperature during solidification. The other techniques compared the results of heat flow simulations with the fluence dependence of the peak melt depth obtained by transient conductance, the fluence dependence of the melt duration determined from time-resolved reflectivity and transient conductance, and the fluence threshold for the initiation of melting. This combination of measurements in conjunction with Rutherford backscattering spectrometry permitted the determination of the solid-liquid interface temperature, velocity and partition coefficient, the latent heat of fusion and T0 for Si-4.5 at. % As and Si-9 at. % As alloys. The values of T0 determined by all four independent methods were consistent, indicating overall agreement between the direct experimental measurements and the analyses based on heat flow simulations. T0 was determined to be 1565±25 K for 4.5 at. % As and 1425±25 K for 9 at. % As. In addition, the enthalpy of fusion was determined to be independent of composition for the range studied. The values obtained in this work are compared with previous measurements.Engineering and Applied Science

Nonequilibrium Partitioning During Rapid Solidification of Si-As Alloys

The velocity dependence of the partition coefficient was measured for rapid solidification of polycrystalline Si-4.5 at% As and Si-9 at% As alloys induced by pulsed laser melting. The results constitute the first test of partitioning models both for the high velocity regime and for non-dilute alloys. The continuous growth model (CGM) of Aziz and Kaplan fits the data well, but with an unusually low diffusive speed of 0.46 m/s. The data show negligible dependence of partitioning on concentration, also consistent with the CGM. The predictions of the Hillert-Sundman model are inconsistent with partitioning results. Using the aperiodic stepwise growth model (ASGM) of Goldman and Aziz, an average over crystallographic orientations with parameters from independent single-crystal experiments is shown to be reasonably consistent with these polycrystalline partitioning results. The results, combined with others, indicate that the CGM without solute drag and its extension to lateral ledge motion, the ASGM, are the only models that fit the data for both solute partioning and kinetic undercooling interface response functions. No current solute drag models can match both partitioning and undercooling measurements.Engineering and Applied Science

Cellular Physiology & Biochemistry / A Swelling-Activated Chloride Current in Microglial Cells is Suppressed by Epac and Facilitated by PKA : Impact on Phagocytosis

Background/Aims: Volume-regulated anion channels (VRACs) are of particular importance in regulating the cell volume (CV) and give rise to the swelling-activated Cl- current (ICl,swell), a main component driving global regulatory volume decrease (RVD) during cell swelling. Because ICl,swell affects numerous CV-regulated processes like migration, we assume that its role is also indispensable for phagocytosis which requires local cell swelling. Noradrenaline (NA) modulates phagocytosis in macrophages and microglial cells, macrophage-related cells in the central nervous system. Therefore we evaluated whether NA modulates ICl,swell and phagocytosis in microglia. Methods: Experiments were performed in murine microglial BV-2 and primary mouse microglial cells. Patch clamp experiments were performed in BV-2 cells using the amphotericin-perforated method to minimize cytosolic disturbances. Phagocytosis was quantified by scanning electron microscopy. Results: Following activation of ICl,swell by a hypotonic bath solution, noradrenaline, as well as the -adrenergic agonist isoproterenol, evoked a transient decrease of ICl,swell. Repeated application of adrenergic agonists caused a decline of this electrical response. Application of the agonist of exchange protein directly activated by cAMP (Epac), 8-pCPT-2-O-Me-cAMP, or the protein kinase A inhibitor H89 caused a persistent suppression of ICl,swell. When isoproterenol was added concomitantly with the hypotonic saline, ICl,swell developed more rapidly compared to control conditions. Uptake of IgG-coated beads was suppressed by NA or H89 when quantified after 15 min of exposure. Conclusion: The activation of -adrenergic receptors in microglial cells triggers a cAMP-Epac-dependent and a cAMP-PKA-dependent cascade which affects phagocytosis via modulation of the swelling-activated Cl- current ICl,swell.(VLID)359693