Purification of biologically active SPARC expressed in Saccharomyces cerevisiae

SPARC (secreted protein, acidic and rich in cysteine) is a secreted, Ca+2-binding glycoprotein that modulates interactions between cells and their immediate extracellular matrix. Traditional sources of SPARC have been mammalian bone, platelets, a basement membrane tumor, and cultured cells; most if not all preparations, however, contain platelet-derived growth factor and one or more serum proteins that bind specifically to purified SPARC. To avoid these contaminants, as well as the toxic lipid moiety associated with endotoxin, we expressed recombinant wild-type and a mutated murine SPARC in two strains of Saccharomyces cerevisiae: one strain was transfected with an expression vector encoding a proprietory signal peptide that directed the secretion of the recombinant protein. Recombinant SPARC was also purified from cell lysates of a different, nonreverting strain of S. cerevisiae that was optimized for large-scale fermentation runs. A mutant murine SPARC lacking the single glycosylation site was also expressed following substitution of Asn98 with Asp98 in the wild-type sequence. Purification of SPARC was achieved by copper-affinity and hydrophobic-interaction chromatography. Both the wild-type and the glycosylation-defective recombinant proteins exhibited high levels of activity in two bioassays with endothelial cells: inhibition of cell spreading/disruption of actin microfilaments and competition for the binding of nonrecombinant 125I-labeled SPARC to the cell surface. The availability of biologically active, recombinant SPARC will facilitate investigation of the structural and functional properties of this protein, which is expressed at high levels in healing wounds, atherosclerotic plaque, and several cancers and diseases of connective tissue.     

Toward an understanding of situational judgment item validity and group differences.

This paper evaluates 2 adjustments to common scoring approaches for situational judgment tests (SJTs). These adjustments can result in substantial improvements to item validity, reductions in mean racial differences, and resistance to coaching designed to improve scores. The first adjustment, applicable to SJTs that use Likert scales, controls for elevation and scatter (Cronbach & Gleser, 1953). This adjustment improves item validity. Also, because there is a White–Black mean difference in the preference for extreme responses on Likert scales (Bachman & O'Malley, 1984), these adjustments substantially reduce White–Black mean score differences. Furthermore, this adjustment often eliminates the score elevation associated with the coaching strategy of avoiding extreme responses (Cullen, Sackett, & Lievens, 2006). Item validity is shown to have a U-shaped relationship with item means. This holds both for SJTs with Likert score response formats and for SJTs where respondents identify the best and worst response option. Given the U-shaped relationship, the second adjustment is to drop items with midrange item means. This permits the SJT to be shortened, sometimes dramatically, without necessarily harming validity. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Molecular mechanisms of vertebrate left-right development

Patterning of all tissues and organs in the vertebrate embryo occurs along the dorsoventral (DV), anteroposterior (AP), and left-right (LR) body axes. Whereas significant progress has been made in identifying the processes underlying DV and AP patterning, relatively little is known about mechanisms guiding LR development. The significant incidence of human disease conditions associated with LR laterality defects, particularly those of the cardiovascular system, underscores the importance of understanding how LR asymmetries become established in the embryo. The focus of this review is on recently identified genes that are involved in generation of vertebrate LR asymmetry, and the proposed cellular and molecular mechanisms by which they might function in initiation, propagation and interpretation of LR patterning information.     

An extension of the dehoff growth path analysis

Growth path analysis is discussed from both deterministic and stochastic points of view. If nucleation is assumed deterministic, the number density function is a solution of the continuity equation. If nucleation is considered a stochastic process, the number density function loses its meaning and must be replaced by a probability density function. Im portant implications of stochastic nucleation are discussed.     

Binary and ternary distillation in a stirred mass transfer cell

A continuous flow stirred cell operating at point conditions has been developed for the study of mass transfer in distillation. Results for the methanol-isopropanol and methanol-ethanol binary systems showed that 85–100% of the resistance to mass transfer resided in the vapour phase, and that the vapour side coefficients correlated well with those obtained in the same cell for gas phase controlled absorption. Flux rates for the ternary system methanol-ethanol-isopropanol were in fair agreement with those predicted from the binary data, using a modified form of the Krishna-Standart theory. It is concluded that the experimental technique shows considerable promise, but requires further development to overcome discrepancies in the interfacial heat balance.     

Lateralization of repeated filtered transients

The catabolic phase of thyroid secretion --thyroglubulin (Tg) uptake and hydrolysis, release of hormones--has been investigated in vitro, by incubating thyroid slices with 0.1% labelled Tg, in cases of isolated adenomas. Twenty-two specimens were examined: 11 cold follicular adenomas, 5 hot nodules from euthyroid patients, 2 untreated toxic adenomas, and 4 pretreated toxic adenomas. The results were compared with those obtained in 11 specimens of normal tissue. Tg pinocytosis (the amount of Tg taken up by the slices per mg of tissue) was severely impaired in all the non-toxic nodules, cold or hot, i.e., whatever the in vivo activity of the thyroid iodide pump. In toxic adenomas, every step of the catabolic hormonogenesis was activated: high pinocytotic activity, increased Tg hydrolysis, and the discharge of hormonal products; in pretreated cases, the whole process was slowed down. Enzymic activity of the acid hydrolases, beta-galactosidase and cathepsin, was elevated in all the nodules so far investigated, particularly in the toxic adenomas.     

A stability analysis of the plate-to-needle morphological transition

The plate-to-needle morphological transition was investigated by means of a stability analysis of a growing platelet. Conditions were found for the evolution of a parallel array of needles from a sinusoidal perturbation of the plate tip. The theory was applied to the Fe-C system using recently obtained kinetic data and the transition region was shown on the Fe-C phase diagram. On the basis of the maximum growth rate postulate the evolving needle spacing was determined by the fastest growing perturbation wavelength. This spacing was plotted vs the dimensionless supersaturation for transformation temperatures of 700°, 650°, and 550°C. A multiple-sectioning technique was used to experimentally determine the morphology of Widmanstätten precipitates in four samples and good agreement was found with theory. Blade or lath-like precipitates, rather than needles, were found to be the result of platelet instability.     

Automated MALDI matrix deposition method with inkjet printing for imaging mass spectrometry

Careful matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is critical for producing reproducible analyte ion signals. Traditional methods for matrix deposition are often considered an art rather than a science, with significant sample-to-sample variability. Here we report an automated method for matrix deposition, employing a desktop inkjet printer (<$200) with 5760 x 1440 dpi resolution and a six-channel piezoelectric head that delivers 3 pL/drop. The inkjet printer tray, designed to hold CDs and DVDs, was modified to hold microscope slides. Empty ink cartridges were filled with MALDI matrix solutions, including DHB in methanol/water (70:30) at concentrations up to 40 mg/mL. Various samples (including rat brain tissue sections and standards of small drug molecules) were prepared using three deposition methods (electrospray, airbrush, inkjet). A linear ion trap equipped with an intermediate-pressure MALDI source was used for analyses. Optical microscopic examination showed that matrix crystals were formed evenly across the sample. There was minimal background signal after storing the matrix in the cartridges over a 6-month period. Overall, the mass spectral images gathered from inkjet-printed tissue specimens were of better quality and more reproducible than from specimens prepared by the electrospray and airbrush methods.     

Fabrication of self-aligned 90-nm fully depleted SOI CMOS SLOTFETs

We have developed a novel sub-100-nm fully depleted silicon-on-insulator (SOI) CMOS fabrication process, in which conventional 248-nm optical lithography and nitride spacer technology are used to define slots in a sacrificial layer (SLOTFET process). This process features a locally thinned SOI channel with raised source-drain regions, and a low-resistance T-shaped poly-Si gate; Both n- and p-channel MOSFETs with 90-nm gate length have been demonstrated. At a 0.5 V bias voltage, ring-oscillator propagation delay of less than 50 ps per stage has been measured