Effects of introduced aspartic and glutamic acid residues on the P'1 substrate specificity, pH dependence and stability of carboxypeptidase Y

Carboxypeptidase Y is a serine carboxypeptidase isolated from Saccharomyces cerevisiae with a preference for C-terminal hydrophobic amino acid residues. In order to alter the inherent substrate specificity of CPD-Y into one for basic amino acid residues in P'1, we have introduced Asp and/or Glu residues at a number of selected positions within the S'1 binding site. The effects of these substitutions on the substrate specificity, pH dependence and protein stability have been evaluated. The results presented here demonstrate that it is possible to obtain significant changes in the substrate preference by introducing charged amino acids into the framework provided by an enzyme with a quite different specificity. The introduced acidic amino acid residues provide a marked pH dependence of the (kcat/Km)FA-A-R-OH/(kcat/Km)FA-A-L-OH ratio. The change in stability upon introduction of Asp/Glu residues can be correlated to the difference in the mean buried surface area between the substituted and the substituting amino acid. Thus, the effects of acidic amino acid residues on the protein stability depend upon whether the introduced amino acid protrudes from the solvent accessible surface as defined by the surrounding residues in the wild type enzyme or is submerged below.     

Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow

The adhesive mechanisms allowing hematopoietic progenitor cells (HPC) homing to the bone marrow (BM) after BM transplantation are poorly understood. We investigated the role of endothelial selectins and vascular cell adhesion molecule-1 (VCAM-1) in this process. Lethally irradiated recipient mice deficient in both P-and E-selectins (P/E-/-), reconstituted with minimal numbers (相似文献   

Expression of beta 2-integrins and L-selectin on polymorphonuclear leukocytes in septic patients

Adhesion molecules on polymorphonuclear leukocytes (PMNL) play an important role in nonspecific defense mechanisms directed at invading microorganisms. When local infection, however, cannot be controlled, a systemic inflammatory response syndrome (SIRS) ensues which may progress to septic shock and multiple organ failure, these being major determinants of the patient's outcome. In the present study, the expression of beta 2-integrins and L-selectin on blood PMNL was measured on subsequent days in patients with sepsis (n = 17) and in healthy volunteers (n = 15). beta 2-Integrins and L-selectin molecules were detected by flow cytometry, using the monoclonal antibodies IB4 (anti-CD18) and Dreg200 (anti-CD62L), respectively. Adhesion molecules were determined at baseline immediately after blood collection and also 45 min after incubation of cells in vitro at body temperature to allow for spontaneous regulation. In addition, PMNL were activated by receptor-dependent and receptor-independent stimuli to characterize stimulus-specific adhesion molecule expression. In parallel with the measurement of adhesion molecules, severity of sepsis was assessed by the Elebute score. The results demonstrate significant differences in the basal, spontaneous and stimulus-induced expression of adhesion molecules between healthy volunteers, survivors (n = 11) and nonsurvivors (n = 6). Moreover, when survivors and nonsurvivors with severe sepsis (Elebute score > 12) were compared, basal expressions of both beta 2-integrins and L-selectin were significantly lower in patients who did not survive. Thus, measurement of adhesion molecules on circulating PMNL may be useful to identify septic patients at high risk for lethal outcome.     

Lidar-based mapping of flood control levees in South Louisiana

Flood protection in south Louisiana is largely dependent on earthen levees, and in the aftermath of Hurricane Katrina the state’s levee system has received intense scrutiny. Accurate elevation data along the levees are critical to local levee district managers responsible for monitoring and maintaining the extensive system of non-federal levees in coastal Louisiana. In 2012, high resolution airborne lidar data were acquired over levees in Lafourche Parish, Louisiana, and a mobile terrestrial lidar survey was conducted for selected levee segments using a terrestrial lidar scanner mounted on a truck. The mobile terrestrial lidar data were collected to test the feasibility of using this relatively new technology to map flood control levees and to compare the accuracy of the terrestrial and airborne lidar. Metrics assessing levee geometry derived from the two lidar surveys are also presented as an efficient, comprehensive method to quantify levee height and stability. The vertical root mean square error values of the terrestrial lidar and airborne lidar digital-derived digital terrain models were 0.038 m and 0.055 m, respectively. The comparison of levee metrics derived from the airborne and terrestrial lidar-based digital terrain models showed that both types of lidar yielded similar results, indicating that either or both surveying techniques could be used to monitor geomorphic change over time. Because airborne lidar is costly, many parts of the USA and other countries have never been mapped with airborne lidar, and repeat surveys are often not available for change detection studies. Terrestrial lidar provides a practical option for conducting repeat surveys of levees and other terrain features that cover a relatively small area, such as eroding cliffs or stream banks, and dunes.     

Design and construction of a high temperature and pressure electrodeless conductivity probe

An electrodeless conductivity probe has been designed and tested at temperatures up to 250 degrees C and pressures of 4.13 MPa (600 psi). A unique pressure seal permits temperature cycling. Design of the electronics permits accurate measurement of conductivities at various elevated temperatures in spite of the change of magnetic properties of the toroids with temperature. The design of the probe can be adapted for insertion, in-line, or bypass operation.     

Discovery of an Allosteric Ligand Binding Site in SMYD3 Lysine Methyltransferase

SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug-like compounds was screened in a biosensor-based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (K_D=42 and 84 μM, respectively). Co-crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C-terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3–HSP90 binding was confirmed (K_D=13 μM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action.     

Accuracy assessment of a mobile terrestrial lidar survey at Padre Island National Seashore

The higher point density and mobility of terrestrial laser scanning (light detection and ranging (lidar)) is desired when extremely detailed elevation data are needed for mapping vertically orientated complex features such as levees, dunes, and cliffs, or when highly accurate data are needed for monitoring geomorphic changes. Mobile terrestrial lidar scanners have the capability for rapid data collection on a larger spatial scale compared with tripod-based terrestrial lidar, but few studies have examined the accuracy of this relatively new mapping technology. For this reason, we conducted a field test at Padre Island National Seashore of a mobile lidar scanner mounted on a sport utility vehicle and integrated with a position and orientation system. The purpose of the study was to assess the vertical and horizontal accuracy of data collected by the mobile terrestrial lidar system, which is georeferenced to the Universal Transverse Mercator coordinate system and the North American Vertical Datum of 1988. To accomplish the study objectives, independent elevation data were collected by conducting a high-accuracy global positioning system survey to establish the coordinates and elevations of 12 targets spaced throughout the 12 km transect. These independent ground control data were compared to the lidar scanner-derived elevations to quantify the accuracy of the mobile lidar system. The performance of the mobile lidar system was also tested at various vehicle speeds and scan density settings (e.g. field of view and linear point spacing) to estimate the optimal parameters for desired point density. After adjustment of the lever arm parameters, the final point cloud accuracy was 0.060 m (east), 0.095 m (north), and 0.053 m (height). The very high density of the resulting point cloud was sufficient to map fine-scale topographic features, such as the complex shape of the sand dunes.     

EFFECT OF COMPOSITION ON PROPERTIES OF CONE 11 ZIRCONIUM SILICATE-OPACIFIED GLAZES *

Studies were made of the effects of magnesium, calcium, barium, strontium, and zinc oxides on the gloss, texture, and opacity of one-fire, zirconium silicate-opacified glazes at cone 11. The opacity was determined with a Hunter multipurpose reflectometer and the gloss was observed visually. Limits for the various fluxes were determined and a practical base glaze formula was developed.