Plasmid transformation of Bacillus sphaericus 1593

Plasmids pUB110 and pBC16 were introduced by protoplast transformation into the entomocidal bacterium Bacillus sphaericus 1593. Transformants expressed the antibiotic resistance determinants present on the plasmid and exhibited sporulation frequencies and larvicidal toxicities which were equivalent to those characteristic of the parent strain. These transformations constitute the first report of genetic transfer in B. sphaericus.     

Evaluation of four methods for computing parameter sensitivities in dynamic system models

Computation of state sensitivities with respect to parameters can be a difficult and costly numerical problem when the number of states and parameters is large, or when sensitivities must be computed repeatedly, as with many optimization algorithms. Four methods are evaluated in terms of solution accuracy, and computer-time and storage requirements: direct numerical integration of the complete sensitivity-system differential equations, a reduced-order method based on the controllable states of the sensitivity system, a numerical-quadratures technique applied directly to the analytic solution of the original system, and an approach based on the solution of the transition matrix. Three linear system models, with four different types of inputs, were used as test cases, the largest having 6 states and 12 parameters. The reduced-order method was the most time-efficient in a majority of cases, but it was prone to numerical instability problems in certain situations which may be encountered in applications. It also had the largest storage requirements. For the highest-order system, only direct numerical integration and the transition-matrix method produced sufficiently accurate results for most applications, because of matrix-inversion problems with the other methods. For impulse inputs, the transition-matrix and the numerical-quadratures methods overall were the most computationally efficient, but the transition-matrix approach required much more memory storage.     

Host selection as a downstream strategy: polyelectrolyte precipitation of beta-glucuronidase from plant extracts.

Host selection can be a strategy to simplify downstream processing for protein recovery. Advancing capabilities for using plants as hosts offers new host opportunities that have received only limited attention from a downstream processing perspective. Here, we investigated the potential of using a polycationic precipitating agent (polyethylenimine; PEI) to precipitate an acidic model protein (beta-glucuronidase; GUS) from aqueous plant extracts. To assess the potential of host selection to enhance the ease of recovery, the same procedure was applied to oilseed extracts of canola, corn (germ), and soy. For comparison, PEI precipitation of GUS was also evaluated from a crude bacterial fermentation broth. Two versions of the target protein were investigated--the wild-type enzyme (WTGUS) and a genetically engineered version containing 10 additional aspartates on each of the enzyme's four homologous subunits (GUSD10). It was found that canola was the most compatible expression host for use with this purification technique. GUS was completely precipitated from canola with the lowest dosage of PEI (30 mg PEI/g total protein), and over 80% of the initial WTGUS activity was recovered with 18-fold purification. Precipitation from soy gave yields over 90% for WTGUS but only 1.3-fold enrichment. Corn, although requiring the most PEI relative to total protein to precipitate (210 mg PEI/g total protein for 100% precipitation), gave intermediate results, with 81% recovery of WTGUS activity and a purification factor of 2.6. The addition of aspartate residues to the target protein did not enhance the selectivity of PEI precipitation in any of the systems tested. In fact, the additional charge reduced the ability to recover GUSD10 from the precipitate, resulting in lower yields and enrichment ratios compared to WTGUS. Compared to the bacterial host, plant systems provided lower polymer dosage requirements, higher yields of recoverable activity and greater purification factors.     

Conformational Space Comparison of GnRH and lGnRH-III using Molecular Dynamics,Cluster Analysis and Monte Carlo Thermodynamic Integration

Abstract

The conformational space available to GnRH and 1GnRH-III was compared using 5.2 ns constant temperature and pressure molecular dynamics simulations with explicit TIP3P solvation and the AMBER v. 5.0 force field. Cluster analysis of both trajectories resulted in two groups of conformations. Results of free energy calculations, in agreement with previous experimental data, indicate that a conformation with a turn from residues 5 through 8 is preferred for GnRH in an aqueous environment. By contrast, a conformation with a helix from residues 2 through 7 with a bend from residues 6 through 10 is preferred for 1GnRH-III in an aqueous environment. The side chains of His² and Trp³ in 1GnRH-III occupy different regions of phase space and participate in weakly polar interactions different from those in GnRH. The unique conformational properties of 1GnRH-III may account for its specific anti cancer activity.     

Time dependence of recruitment and derecruitment in the lung: a theoretical model.

Recruitment and derecruitment (R/D) of air spaces within the lung is greatly enhanced in lung injury and is thought to be responsible for exacerbating injury during mechanical ventilation. There is evidence to suggest that R/D is a time-dependent phenomenon. We have developed a computer model of the lung consisting of a parallel arrangement of airways and alveolar units. Each airway has a critical pressure (Pcrit) above which it tends to open and below which it tends to close but at a rate determined by how far pressure is from Pcrit. With an appropriate distribution of Pcrit and R/D velocity characteristics, the model able to produce realistic first and second pressure-volume curves of a lung inflated from an initially degassed state. The model also predicts that lung elastance will increase transiently after a deep inflation to a degree that increases as lung volume decreases and as the lung becomes injured. We conclude that our model captures the time-dependent mechanical behavior of the lung due to gradual R/D of lung units.