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.     

Rapid infusion system for neurosurgical treatment of massive intraoperative hemorrhage

Using an illustrative case of severe closed head injury that resulted in a posterior fossa epidural hematoma (EDH) and supratentorial epidural/subdural hematomas (SDH), the massive blood losses associated with operative repair of the torn sigmoid sinus and the significant fluid losses associated with refractory diabetes insipidus were treated by the intraoperative use of the Rapid Infusion System (RIS, Haemonetics). The RIS can rapidly infuse warm blood, crystalloid, or colloid at rates up to 1.5 L/min, thereby limiting the commonly associated hypotension, hypothermia, and coagulopathies. During the suboccipital craniectomy for evacuation of the EDH and repair of the sigmoid sinus, the patient required 18 units of blood replacement secondary to a large tear in the sigmoid sinus. During a separate craniotomy for evacuation of the SDH, the patient also developed diabetes insipidus, which increased the operative fluid replacement to 39 L. Despite these massive blood and fluid losses, the RIS limited the hypotension to less than 2 min and prevented hypothermia and the frequently associated coagulopathies. When used in a neurosurgical setting associated with massive blood and/or fluid losses, the RIS accomplishes three important objectives: (1) rapid infusion of intravenous fluids for maintaining perfusion pressure, (2) rapid warming of fluids despite high intravenous infusion rates of cold crystalloids, thereby preventing intraoperative hypothermia, and (3) continuous monitoring of infusion rates and totals.     

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   

Ultrasonically activated shears in thyroid surgery

BACKGROUND: Ultrasonically activated shears (UAS) have been documented to be both safe and fast devices in laparoscopic surgery. We studied whether the use of UAS would have some advantage in thyroid surgery. METHODS: Thyroidectomies, performed by one senior endocrine surgeon between December 1996 and February 1997, were retrospectively matched, with patients operated on by the same surgeon using the conventional method. RESULTS: Six pairs of total thyroidectomies and one pair of lobectomies could be matched. Mean operating time was 100 minutes for the patients operated on with the UAS and 154 minutes for the patients operated on with the conventional method. The mean operating time with the UAS was thus on average 64.6% of the operation time with the conventional method, with a 95% confidence interval from 50.1% to 83.5% (t = 4.00, 6 df, P = 0.007). CONCLUSIONS: In this material the use of UAS reduced significantly operating time in thyroidectomies.     

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.     

Large Particle Penetration During PM10 Sampling

The objective of the present study was to characterize the performance of a federal reference method (FRM) PM₁₀ size-selective inlet using analysis methods designed to minimize uncertainty in measured sampling efficiencies for large particles such as those most often emitted from agricultural operations. The performance of an FRM PM₁₀ inlet was characterized in a wind tunnel at a wind speed of 8 km/h. Data were also collected for 20 and 25 μm particles at wind speeds of 2 and 24 km/h. Results of the present sampler evaluation compared well with those of previous studies for a similar inlet near the cutpoint, and the sampler passed the criteria required for certification as a FRM sampler when tested at 8 km/h. Sampling effectiveness values for particles with nominal diameters of 20 and 25 μm exceeded 3% for 8 and 24 km/h wind speeds in the present study and were statistically higher than both the “ideal” PM₁₀ sampler (as defined in 40 CFR 53) and the ISO (1995) standard definition of thoracic particles (p < 0.05) for 25 μm particles leading to the potential for significant sampling bias relative to the “ideal” PM₁₀ sampler when measuring large aerosols.

Copyright 2014 American Association for Aerosol Research     

Single fundamental-mode output power exceeding 6 mW from VCSELs with a shallow surface relief

A single fundamental-mode output power of 6.5 mW was achieved from an 850-nm vertical-cavity surface-emitting laser (VCSEL) with a shallow surface relief, the highest single-mode power ever reported using this technique. The VCSELs were fabricated from epitaxial material grown to yield an antiphase reflection from the topmost layer. A circular surface relief, acting as a mode discriminator, was etched in the center to reduce the mirror loss for the fundamental mode. This "inverted" surface-relief technique offers relaxed etch depth control and, therefore, improves reproducibility and yield.     

Design and evaluation of fundamental-mode and polarization-stabilized VCSELs with a subwavelength surface grating

We demonstrate 850-nm oxide-confined vertical-cavity surface-emitting lasers (VCSELs) with a locally etched subwavelength surface grating that are single-mode and polarization stable from threshold up to thermal roll-over, reaching /spl sim/4 mW of output power. The side-mode suppression ratio (SMSR) is >30 dB and the orthogonal polarization suppression ratio (OPSR) is /spl sim/20 dB. Moreover, no distortion of the far-field beam profile is observed as a result of the surface grating. Our numerical calculations show that a carefully designed VCSEL can have a high simultaneous mode and polarization selectivity without a significant increase in loss for the favored fundamental mode with polarization state perpendicular to the grating lines. This indicates characteristics such as threshold current and resonance frequency will not be notably degraded. The calculations also show a low sensitivity to variations in grating etch depth and duty cycle, which relaxes fabrication tolerances. In our experimental parametric study, where the oxide aperture diameter, surface grating diameter, and grating duty cycle were varied, the combined mode and polarization selection was investigated. For an optimum combination of oxide aperture and surface grating diameters of 4.5 and 2.5 /spl mu/m, respectively, the device is found to be single-mode and polarization stable for a broad range of grating duty cycles, from 55% to 75%, with only a small variation in other laser performances, which is in line with theory.