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.     

Muscle transformation of the sartorius muscle in a canine model: clinical impact for electrodynamic graciloplasty as a "neosphincter"

PURPOSE: Transformation of fast-twitching skeletal muscles to slow-twitching, slowly fatigable muscles has become of clinical interest in the recent past. Transposition and transformation of the gracilis muscle to use it as a substitute for a resected or defected anal sphincter (graciloplasty) have been reported as achieving promising results in the treatment of fecal incontinence caused by sphincter defects or following abdominoperineal anorectal excision for cancer. METHOD: This experimental study used a canine model and the sartorius muscle to evaluate the functional efficiency of two different configurations of the muscle loop to compare the presently applied transformation program (8 weeks) with a shorter (5 weeks) protocol. In six beagle dogs, both sartorius muscles were wrapped around two stomas, either in an alpha fashion or in the so-called split-sling technique. Muscle transformation was achieved by controlled neuromuscular stimulation either during eight (Program A) or five weeks (Program B). After completion of the transformation period, the function of the muscle slings was evaluated by manometry, and histomorphologic evaluation of the sartorius muscles was performed. RESULTS: It was shown that muscle transformation led to a slowly fatigable muscle that made it possible to perform continuos (tetanic) contraction, regardless of the configuration or the duration of the transformation. Median pressures created by these muscles also did not differ significantly. In accordance with these functional findings, the histologic evaluation showed the typical, significant increase of Type I fibers in both muscle slings and following both transformation protocols. Although the decrease of fast-twitching Type II fibers was more pronounced following the conventional (8 weeks) program, this finding did not influence the functional results. CONCLUSIONS: Results of our experiment indicate the possibility for using a shorter transformation protocol for transformation of the gracilis muscle during graciloplasty in the clinical setting. Furthermore, the efficacy and safety of the modified (split-sling) wrap technique was demonstrated.     

Determination of dislocation densities in HCP metals from X-ray diffraction line-broadening analysis

X-Ray diffraction (XRD) line-broadening analysis has been performed on highly textured Zr-2.5Nb specimens which had been deformed in tensile tests to produce well-controlled dislocation structures. An iterative deconvolution method has been applied to extract the broadening function for the material, using as standards, a Zr single crystal and a 0 pct deformed specimen. In both cases, for specific tensile tests, a significant contribution to the basal line braodening was observed, which was clearly not directly related to the dislocation structure generated by the deformation, i.e., so-called c-component dislocations having a component of their Burgers vectors perpendicular to the basal plane. Calculations showed that the extent of basal line broadening cannot be attributed to the secondary effect of strain from a-type dislocations, i.e., dislocations with Burgers vectors parallel with the basal plane. It is concluded that most of the line broadening observed was the result of intergranular strain distributions. These distributions are most prominent for grains oriented with their c-axes perpendicular to the tensile-deformation axis and resulted in basal-plane line broadening even when there were few, if any, c-component dislocations present. This article is based on a presentation made in the symposium entitled “Defect Properties and Mechanical Behavior of HCP Metals and Alloys” at the TMS Annual Meeting, February 11–15, 2001, in New Orleans, Louisiana, under the auspices of the following ASM committees: Materials Science and Critical Technology Sector, Structural Materials Division, Electronic, Magnetic & Photonic Materials Division, Chemistry & Physics of Materials Committee, Joint Nuclear Materials Committee, and Titanium Committee.     

Microstructures formed by spray freezing of food fats

The spray-freezing of two food fats, tripalmitin (PPP) and cocoa butter (CB) and mixtures thereof, has been modeled experimentally using a novel single droplet freezing apparatus configured so that temperature profiles or samples for microstructure analysis can be obtained. For 2 mm diameter droplets suspended in a cold air flow at temperatures around 2–15°C, initial cooling rates were on the order of 10 K s⁻¹ and the temperature profiles could be correlated directly to DSC data collected at 20 K min⁻¹, indicating that minimal supercooling of the materials occurred in the droplet form. Microstructure analysis confirmed that PPP crystallized preferentially in mixtures, and that the surface structure was very sensitive to storage conditions. The bulk structure was much less sensitive, and the internal microstructure of the PPP droplets revealed distinct nucleation sites, which were absent from the CB: These persisted in the mixtures up to 50 wt%. X-ray analysis indicated that the fats crystallized in their more stable forms, namely, β for PPP and Form V/V1 in CB.     

Netted radar sensitivity and ambiguity

The performance of netted radar is evaluated in terms of a netted version of the radar equation and the ambiguity function. A software simulation has been developed to allow assessment of netted radar sensitivity and ambiguity properties in both two-and three-dimensional space. Simulation results demonstrate that coherent netted radar sensitivity depends not only on the radar parameters, but also on the numbers, locations and specification of transmit and receive nodes. The netted radar ambiguity function is affected by a combination of many factors. In addition to those that determine the form of the monostatic ambiguity function, the locations of transmit and receive nodes as well as the position and velocity of the target all are shown to play an important role in evaluating system performance. As a result of these dependencies, it is shown that there is severe degradation of range and Doppler resolution when a target crosses sufficiently close to a transmit-receive baseline.     

Prostaglandin synthase-1 and prostaglandin synthase-2 are coupled to distinct phospholipases for the generation of prostaglandin D2 in activated mast cells

Aggregation of IgE cell surface receptors on MMC-34 cells, a murine mast cell line, induces the synthesis and secretion of prostaglandin D2 (PGD2). Synthesis and secretion of PGD2 in activated MMC-34 cells occurs in two stages, an early phase that is complete within 30 min after activation and a late phase that reaches a maximum about 6 h after activation. The early and late phases of PGD2 generation are mediated by prostaglandin synthase 1 (PGS1) and prostaglandin synthase 2 (PGS2), respectively. Arachidonic acid, the substrate for both PGS1 and PGS2, is released from membrane phospholipids by the activation of phospholipases. We now demonstrate that in activated mast cells (i) secretory phospholipase A2 (PLA2) mediates the release of arachidonic acid for early, PGS1-dependent synthesis of PGD2; (ii) secretory PLA2 does not play a role in the late, PGS2-dependent synthesis of PGD2; (iii) cytoplasmic PLA2 mediates the release of arachidonic acid for late, PGS2-dependent synthesis of PGD2; and (iv) a cytoplasmic PLA2-dependent step precedes secretory PLA2 activation and is necessary for optimal PGD2 production by the secretory PLA2/PGS1-dependent early pathway.     

Beta-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo

Experimental glomerulonephritis was induced in rats to investigate the consequence of the antigen-antibody interaction on the surface of glomerular endothelial cells (GENs). A lectin, Lens culinaris hemoagglutinin (LCH), was first planted in the left kidney by isolated perfusion of a left kidney, and then the circulation was reestablished. Rabbit anti-LCH antibodies were injected from the tail vein 3 minutes after the recirculation of the left kidney, and acute glomerulonephritis ensued. Fifteen minutes after the injection, rabbit immunoglobulin G (IgG), rat C3, and LCH were observed exclusively on the surface of GENs. Accumulation of platelets was prominent. Three hours later, the immune deposits were seen in the subendothelial space, and the polymorphonuclear cells were the dominant infiltrate in the glomeruli. Up to the seventh day, immune deposits were seen in the subendothelial space, and the widening of this area was increasingly observed. Fourteen days later, immune deposits containing rat IgG were observed in the subepithelial area, but they were only occasionally seen in the subendothelial space and in the mesangial area. No crescent formation was seen at day 14, but the mesangial area was expanded, with an increased number of cells. The number of nuclei in the cross-section of a glomerulus increased after the induction of glomerulonephritis, but the number of leukocyte common antigen-positive cells (infiltrating cells) decreased gradually from day 4 to day 14. The staining of Thy-1.1, a marker of mesangial cell, was markedly enlarged in the glomerulus at day 14. These data suggest that mesangial proliferative glomerulonephritis can be induced by the antigen-antibody interaction on the surface of GENs.     

Properties of permease dimer, a fusion protein containing two lactose permease molecules from Escherichia coli

An engineered fusion protein containing two tandem lactose permease molecules (permease dimer) exhibits high transport activity and is used to test the phenomenon of negative dominance. Introduction of the mutation Glu-325-->Cys into either the first or the second half of the dimer results in a 50% decrease in activity, whereas introduction of the mutation into both halves of the dimer abolishes transport. Lactose transport by permease dimer is completely inactivated by N-ethylmaleimide; however, 40-45% activity is retained after N-ethylmaleimide treatment when either the first or the second half of the dimer is replaced with a mutant devoid of cysteine residues. The observations demonstrate that both halves of the fusion protein are equally active and suggest that each half may function independently. To test the possibility that oligomerization between dimers might account for the findings, a permease dimer was constructed that contains two different deletion mutants that complement functionally when expressed as untethered molecules. Because this construct does not catalyze lactose transport to any extent whatsoever, it is unlikely that the two halves of the dimer interact or that there is an oligomeric interaction between dimers. The approach is consistent with the contention that the functional unit of lactose permease is a monomer.     

Drugs, memory, and metamemory: A dose-effect study with lorazepam and scopolamine.

This experiment was designed to use the graded dose-related amnesia produced by the benzodiazepine lorazepam (1.0, 2.0 mg/70 kg, oral) and the anticholinergic scopolamine (0.3, 0.6 mg/70 kg, subcutaneous) as a tool to explore the cognitive and neurochemical mechanisms underlying metamemory in the judgment of learning paradigm, with a placebo-controlled independent groups design in healthy volunteers (n=12/group). Results provide evidence for a pharmacological dissociation between effects on memory versus metamemory (relative accuracy of item-by-item monitoring) across a range of levels of memory performance and suggest that the drugs selectively impair those aspects of metamnemonic monitoring that require participants' awareness of their overall current state of functioning (absolute accuracy of prospective item-by-item monitoring, prospective global monitoring) but not those that rely solely on assessment of individual item characteristics (relative accuracy of item-by-item monitoring). (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A method to study the history of a double oxide film defect in liquid aluminum alloys

Entrained double oxide films have been held responsible for reductions in mechanical properties in aluminum casting alloys. However, their behavior in the liquid metal, once formed, has not been studied directly. It has been proposed that the atmosphere entrapped in the double oxide film defect will continue to react with the liquid metal surrounding it, perhaps leading to its elimination as a significant defect. A silicon-nitride rod with a hole in one end was plunged into liquid aluminum to hold a known volume of air in contact with the liquid metal at a constant temperature. The change in the air volume with time was recorded by real-time X-ray radiography to determine the reaction rates of the trapped atmosphere with the liquid aluminum, creating a model for the behavior of an entrained double oxide film defect. The results from this experiment showed that first oxygen, and then nitrogen, was consumed by the aluminum alloy, to form aluminum oxide and aluminum nitride, respectively. The effect of adding different elements to the liquid aluminum and the effect of different hydrogen contents were also studied. This article is based on a presentation made in the John Campbell Symposium on Shape Casting, held during the TMS Annual Meeting, February 13–17, 2005, in San Francisco, CA.