Construction and optimization of a CC49-based scFv-beta-lactamase fusion protein for ADEPT

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).     

Spatial filter pinhole for high-energy pulsed lasers

Spatial filters are essential components for maintaining high beam quality in high-energy pulsed laser systems. The long-duration (21 ns) high-energy pulses envisioned for future inertial-confinement fusion drive systems, such as the U.S. National Ignition Facility (NIF), are likely to lead to increased plasma generation and closure effects within the pinholes in the spatial filters. The design goal for the pinhole spatial filter for the NIF design is to remove small-angle scatter in the beam to as little as a ?100-murad divergence. It is uncertain whether this design requirement can be met with a conventional pinhole design. We propose a new pinhole architecture that addresses these issues by incorporating features intended to reduce the rate of plasma generation. Initial experiments with this design have verified its performance improvement relative to a conventional pinhole design.     

A path analysis of factors associated with distress among first-degree female relatives of women with breast cancer diagnosis.

Patterns and predictors of psychological distress in first-degree female relatives (N = 624) of newly diagnosed breast cancer patients were explored. First-degree female relatives who were high monitors reported greater cancer-specific and general distress than did low monitors. Greater optimism was associated with lower cancer-specific distress. Optimism's effect on general distress was moderated by women's level of monitoring. Greater optimism was associated with lower general distress for both high and low monitors, but the effect was stronger for high monitors than for low monitors. Avoidance and engaged coping were associated with higher distress. A close relationship with the cancer patient was related to higher cancer-specific distress but lower general distress. Further understanding of the process of adjustment in these women awaits longitudinal study. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Metabolism of the antiandrogenic drug (Flutamide) by human CYP1A2

The antiandrogenic drug, flutamide, is widely used in the treatment of carcinoma of the prostate. The present study examines the metabolism of flutamide by human liver microsomes and purified recombinant human cytochrome P450s (CYP), expressed as fusion proteins. These studies show the principal role of CYP1A2 in the metabolism of flutamide to 2-hydroxyflutamide. A minor metabolite is formed during the metabolism of flutamide by CYP3A4 in the presence of an excess of added purified NADPH-P450 reductase. The metabolism of flutamide is inhibited by low concentrations of alpha-naphthoflavone and ketoconazole. Other substrates of CYP1A2, such as phenacetin, imipramine, caffeine, and estradiol, are also inhibitors of flutamide metabolism by CYP1A2. Of interest is the inhibition of flutamide metabolism by its metabolite, 2-hydroxyflutamide, and the inhibition of the 2- and 4- hydroxylation of estradiol by flutamide. CV1 cells do not metabolize flutamide to 2-hydroxyflutamide. In assays performed using this cell line transfected with the cDNA for the androgen receptor, flutamide is a pure antagonist, and 2-hydroxyflutamide, while a more potent androgen receptor (AR) antagonist, activates the AR at higher concentrations. Stable expression of CYPIA2 in these CV1 cells causes flutamide to exhibit agonistic properties at higher concentrations, a behavior not exhibited by cells stably transfected only with the expression vector encoding the AR. These findings raise the possibility that increased conversion of flutamide to 2-hydroxyflutamide or accumulation of 2-hydroxyflutamide in cells may contribute to the anomalous responses to flutamide that are observed in some advanced prostate cancers.     

Anti-Gal binds to pili of Neisseria meningitidis: the immunoglobulin A isotype blocks complement-mediated killing

alpha 1,3-Galactosyl antibodies (anti-Gal) are ubiquitous natural human serum and secretory polyclonal antibodies that bind to terminal galactose-alpha 1,3-galactose (alpha-galactosyl) residues. Serum immunoglobulin G (IgG) anti-Gal can block alternative complement pathway-mediated lysis of representative gram-negative enteric bacteria that bind it to lipopolysaccharide alpha-galactosyl structures, thereby promoting survival of such bacteria in the nonimmune host. We wanted to know whether anti-Gal also could bind to the lipooligosaccharides (LOS) of Neisseria meningitidis. To our surprise, we found that serum and secretory anti-Gal bound to pili but not to LOS of certain strains. This suggested the presence of an immunogenic pilus carbohydrate epitope. Mild periodate oxidation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated outer membrane preparations from strains that bound anti-Gal followed by labeling of the neoaldehyde groups resulted in the labeling of bands that corresponded to pilin and LOS, confirming that pilin contains carbohydrate structures. A Bandeiraea simplicifolia lectin that also binds terminal alpha 1,3-galactosyl residues also bound to pilin. Serum IgG, IgA, and IgM anti-Gal as well as colostral secretory IgA anti-Gal bound to pilin, as judged by immunoblotting, and to the pili of intact piliated organisms, as judged by immunoelectron microscopy. Total serum anti-Gal (IgG, IgA, and IgM) and purified serum IgA1 anti-Gal, but not its purified IgG isotype, blocked complement-mediated lysis of a piliated meningococcal strain that bound anti-Gal to its pili. Colostral anti-Gal secretory IgA blocked killing of the same strain. Thus, anti-Gal IgA may promote disease when it binds to the pili of N. meningitidis strains.     

Development of deep-ocean work system

The Navy is developing a deep-ocean work system comprised of manipulators and a variety of hydraulic tools integrated to accomplish meaningful work tasks to ocean depths of 20,000 ft. The system can readily adapted to the manned submersibles ALVIN, SEACLIFF, and TURTLE, and the robot vehicles CURV III and RUWS, to extend their work capability. In addition, it can be positioned and controlled by divers or operated by itself from a surface support ship. The system was designed to perform a complete work operation on the sea floor without the necessity of resurfacing for tool interchange. Potential tasks include recovery, construction, installation, and repair operations. Low-light level TV cameras, with quartz-iodide flood lights, augment the vehicle operator's vision for tool exchange and work operations.