Purification and characterization of two forms of urokinase

Affinity chromatography on agmatine-Sepharose was used for the separation of two active forms of urokinase (EC 3.4.99.26) from partially purified human urinary urokinase. The approximate molecular weight of the heavier form was 47 000 and of the lighter 33 400. Both forms were homogeneous by sodium dodecyl sulfate gel electrophoresis and by 3H-labeled diisopropylphosphorofluoridate and 14C-labeled p-nitrophenyl-p'-guanidinobenzoate incorporation studies. The 33 400 mol. wt. form had a single chain, and the 47 000 mol. wt. form had two chains (33 100 and 18 600 mol. wt.) linked by disulfide bonds. The specific activity of the heavier form was 104 000 CTA units/mg protein, compared with 226 000 units/mg for the lighter form but the activities per mmol of active site (molar activities) of the two forms were almost identical (9.6-10(9) and 10.2-10(9) CTA units/mmol). Isoelectric focusing on gels showed that the 47 000 material contained one major subform with a pI of 8.60 and a minor subform with a pI of 8.90, while the 33 400 material had three major subforms with pI values of 8.35, 8.60 and 8.70, respectively, and a minor subform with a pI of 8.05. 3H-labeled diisopropylphosphorofluoridate incorporation studies revealed an active-site serine residue in the heavy chain.     

Dexamethasone 21-mesylate: an affinity label of glucocorticoid receptors from rat hepatoma tissue culture cells

We recently described the biological properties of an alpha-keto mesylate derivative of cortisol, cortisol-Mes. Cortisol-Mes exhibited long-term antiglucocorticoid activity, but there was no firm evidence that this activity was irreversible or receptor-mediated. Here we report that dexamethasone mesylate (Dex-Mes), which is the alpha-keto mesylate derivative of the more active glucocorticoid dexamethasone, is a candidate for a steroid-specific affinity label of glucocorticoid receptors. Dex-Mes is relatively stable, like cortisol-Mes, but possesses greater whole-cell antiglucocorticoid activity. However, Dex-Mes also possesses partial agonist activity, which is expressed at somewhat higher concentrations of Dex-Mes than the antagonist activity. Dex-Mes is more efficient than cortisol-Mes in competing for dexamethasone binding to glucocorticoid receptors. Furthermore, Dex-Mes is effective at lower concentrations than cortisol-Mes in causing long-term apparently irreversible antiglucocorticoid effects in whole and broken cells. The cell-free effect of Dex-Mes is specifically prevented by coincubation with an excess of cortisol. These facts argue that the apparently irreversible effects of Dex-Mes are steroid mediated. [3H]Dex-Mes has been used to identify a glucocorticoid-specific, covalently labeled fraction on sodium dodecyl sulfate/polyacrylamide gels with a molecular weight of approximately 85,000. Thus Dex-Mes appears to have been established as an affinity label for glucocorticoid receptors.     

Matched-pair cohort methods in traffic crash research

Standard analysis of matched-pair cohort data requires information only from pairs in which at least one had the study outcome. This can be useful in traffic fatality studies of characteristics that can vary among vehicle occupants, such as seat belt use, as crash databases often lack information about vehicles in which all survived. However, matching crash victims who were in the same vehicle does not necessarily eliminate confounding by vehicle or crash related factors, because the matched occupants must be in different seat positions. This paper reviews three methods for estimating relative risks in matched-pair crash data. The first, Mantel-Haenszel stratified methods, may produce biased estimates if seat position is associated with the outcome. The second, the double-pair comparison method, was designed to deal with confounding by seat position. If the effects of seat position vary according to some vehicle or crash characteristic which is associated with the study exposure, adjustment for this characteristic may be needed to produce unbiased estimates. Third, conditional Poisson regression and Cox proportional hazards regression can produce unbiased estimates, but may require model interaction terms between seat position and vehicle or crash characteristics. This paper reviews some of the strengths and limitations of each of these methods, and illustrates their use in simulated and real crash data.     

Faceted design of channels for low-dispersion electrokinetic flows in microfluidic systems

A novel methodology for designing microfluidic channels for low-dispersion, electrokinetic flows is presented. The technique relies on trigonometric relations that apply for ideal electrokinetic flows, allowing faceted channels to be designed using common drafting software and a hand calculator. Flows are rotated and stretched along the abrupt interface between adjacent regions having differing specific permeability--a quantity with dimensions of length that we introduce to derive the governing equations. Two-interface systems are used to eliminate hydrodynamic rotation of bands injected into channels. Regions bounded by interfaces form faceted flow "prisms" with uniform velocity fields that can be combined with other prisms to obtain a wide range of turning angles and expansion ratios. Lengths of faceted prisms can be varied arbitrarily, simplifying chip layout and allowing the ability to reduce unwanted effects such as transverse diffusion and Joule heating for a given faceted prism. Designs are demonstrated using two-dimensional numerical solutions of the Laplace equation.     

Dielectrophoresis in microchips containing arrays of insulating posts: theoretical and experimental results

Dielectrophoresis (DEP), a nonlinear electrokinetic transport mechanism, can be used to concentrate and sort cells, viruses, and particles. To date, microfabricated DEP-based devices have typically used embedded metal electrodes to apply spatially nonuniform, time-varying (AC) electric fields. We have developed an alternative method in which arrays of insulating posts in a channel of a microchip produce the spatially nonuniform fields needed for DEP. Electrodes may be located remotely, allowing operation of the device down to zero frequency (DC) without excessive problems of electrolysis. Applying a sufficiently large electric field across an insulating-post array produces two flow regimes through a competition between electrokinetic flow (combined electrophoresis and electroosmosis) and dielectrophoresis. "Streaming DEP" is observed when DEP dominates diffusion but is overcome by electrokinetic flow. Particles concentrated by DEP forces in areas of electric field extrema travel electrokinetically down the array in flowing streams. In an array of posts, dielectrophoretic forcing within repeated rows adds coherently to produce flowing streams of highly concentrated and rarefied particles. We demonstrate that this reinforcement is a strong function of alignment of the array with respect to the applied electric field and that the particle concentrations can be "enhanced" or "depleted" along columns of posts, enabling a novel class of continuous-flow, selective particle filter/concentrator devices. To our knowledge, this is the first observation of streaming dielectrophoresis. The second regime is "trapping DEP," in which DEP forces dominate over both diffusion and electrokinetic flow, reversibly immobilizing particles on the insulating posts, enabling inexpensive and embedded batch filter/concentrator devices. Devices can be biased electrically to manipulate particles selectively by varying the field strength to vary the relative magnitudes of electrokinetic flow and DEP. Post shapes are contoured easily to control electric field gradients and, hence, DEP behavior. Simple simulations based on similitude of fluid flow and electric field that solve the Laplace equation to obtain fluid velocity have also been developed to predict the dielectrophoretic behavior in an array of posts. These simulations are in excellent agreement with the experimental observations and provide insight into electrokinetic behavior to enable design of dielectrophoretic concentrators and sorters.     

Composite carbon paste biosensor for phenolic derivatives based on in situ electrogenerated polypyrrole binder

Amperometric biosensors based on new composite carbon paste (CPE) electrodes have been designed for the determination of phenolic compounds. The composite CPEs were prepared by in situ generation of polypyrrole (PPy) within a paste containing the enzyme polyphenol oxidase (PPO). The best paste composition (enzyme/pyrrole monomer/carbon particles/Nujol) was determined for a model enzyme, glucose oxidase, according to the enzymatic activity of the resulting electrodes and to the enzyme leakage from the paste during storage in phosphate buffer. The in situ electrogenerated PPy enables improvement in enzyme immobilization within the paste since practically no enzyme was lost in solution after 72 h of immersion. Moreover, the enzyme activity remains particularly stable under storage since the biocomposite structure maintains 80% of its activity after 1-month storage. Following the optimization of the paste composition, PPO-based carbon paste biosensors were prepared and presented excellent analytical properties toward catechol detection with a sensitivity of 4.7 A M(-1) cm(-2) and a response time lower than 20 s. The resulting biosensors were finally applied to the determination of epicatechin and ferulic acid as flavonol and polyphenol model, respectively.     

Nonequilibrium molecular dynamics calculation of the shear viscosity of carbon dioxide

Nonequilibrium molecular dynamics calculations of the shear viscosity of supercritical carbon dioxide along the 313 K isotherm are reported. Three different intermolecular potential models of increasing complexity are considered: a spherically symmetric Lennard-Jones potential, a two-site Lennard-Jones potential, and a three-site potential which includes a quadrupole-quadrupole moment. Results for the three potentials are compared with experimental data.Paper presented at the Tenth Symposium on Thermophysical Properties, June 20–23, 1988, Gaithersburg, Maryland, U.S.A.     

Conceptualizations of novice and experienced counselors.

We examined the conceptualizations of 23 (11 experienced and 12 novice) counselors, with respect to general counseling process and specific client concerns by means of a two-part conceptual mapping task (CMT). Analyses of the CMT data revealed an interaction effect of counselor experience and generality of conceptual task on the extensiveness of counselor conceptualizations. Experienced counselors displayed slightly more extensive conceptualizations on the general CMT and less extensive conceptualizations on the specific CMT. This interaction effect was interpreted as evidence that experienced counselors possess extensive abstract, general knowledge of counseling that they use to conceptualize specific instances of counseling efficiently and parsimoniously. On the other hand, novice counselors, who lack such abstracted knowledge, engage in more extensive, unique conceptual work for each separate client. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Liz1p, a novel fission yeast membrane protein, is required for normal cell division when ribonucleotide reductase is inhibited

Ribonucleotide reductase activity is required for generating deoxyribonucleotides for DNA replication. Schizosaccharomyces pombe cells lacking ribonucleotide reductase activity arrest during S phase of the cell cycle. In a screen for hydroxyurea-sensitive mutants in S. pombe, we have identified a gene, liz1(+), which when mutated reveals an additional, previously undescribed role for ribonucleotide reductase activity during mitosis. Inactivation of ribonucleotide reductase, by either hydroxyurea or a cdc22-M45 mutation, causes liz1(-) cells in G2 to undergo an aberrant mitosis, resulting in chromosome missegregation and late mitotic arrest. liz1(+) encodes a 514-amino acid protein with strong similarity to a family of transmembrane transporters, and localizes to the plasma membrane of the cell. These results reveal an unexpected G2/M function of ribonucleotide reductase and establish that defects in a transmembrane protein can affect cell cycle progression.