Paul Todd

Application of osmotic dewatering to the controlled crystallization of biological macromolecules and organic compounds

Abstract Several methods of crystallization of biological macromolecules depend upon the transport of water through the vapor phase - a process that is sensitive to ambient conditions (temperature, relative humidity). Other methods depend on the transport of solute by diffusion or through a membrane. By regulating the solute concentration on the outside of a reverse-osmosis membrane it is possible to control the rate at which macromolecules and other solutes are concentrated inside a membrane-bound fluid. The effect of dewatering rate on lysozyme crystal quality and growth rate was assessed. A 3-fold increase in concentration over a 9 day period yielded tetragonal crystals 0.5 mm on a side with sharp edges and with ordering at least to 1.73 A. Transparent crystals of triglycine sulfate were grown by osmotic dewatering; in this case crystal growth could be enhanced or reversed by manipulating the external solution.

Physical effects at the cellular level under altered gravity conditions.

Several modifications of differentiated functions of animal cells cultivated in vitro have been reported when cultures have been exposed to increased or decreased inertial acceleration fields by centrifugation, clinorotation, and orbital space flight. Variables modified by clinorotation conditions include inertial acceleration, convection, hydrostatic pressure, sedimentation, and shear stress, which also affect transport processes in the extracellular chemical environment. Autocrine, paracrine and endocrine substances, to which cells are responsive via specific receptors, are usually transported in vitro (and possibly in certain embryos) by convection and in vivo by a circulatory system or ciliary action. Increased inertial acceleration increases convective flow, while microgravity nearly abolishes it. In the latter case the extracellular transport of macromolecules is governed by diffusion. By making certain assumptions it is possible to calculate the Peclet number, the ratio of convective transport to diffusive transport. Some, but not all, responses of cells in vitro to modified inertial environments could be manifestations of modified extracellular convective flow.

Electrokinetic Demixing of Two-Phase Aqueous Polymer Systems. II. Separation Rates of Polyethylene Glycol–Maltodextrin Mixtures

Abstract Aqueous two-phase extraction techniques have been successfully applied to the purification of enzymes and cells. However, due to their similar physical properties, immiscible aqueous phases do not separate rapidly. A method for enhanced demixing of aqueous two-phase systems in a thermostated vertical electrophoresis column was therefore studied. The effects of the electric field strength, field polarity, temperature, phase composition, and buffer concentration on demixing rates of a polyethylene glycol-maltodextrin (PEG-MDX) system were quantitatively measured. At normal electrical polarity (anode at the top of the column), using a maximum practicable field strength of 26.4 V/cm, the demixing rate was twice that in zero electric field at 25 ± 2°C. With poiarity reversed (anode at the bottom, electric field opposing gravitational settling) at a field of 26.4 V/cm, demixing was 5.5 times as fast as in zero field. Reduction of the temperature from 25 to 14°C caused an increase in demixing rate in th...

Investigations on gel forming media for use in low gravity bioseparations research

Microgravity research includes investigations designed to gain insight on methods of separating living cells. During a typical separation certain real-time measurements can be made by optical methods, but some materials must also be subjected to subsequent analyses, sometimes including cultivation of the separated cells. In the absence of on-orbit analytical or fraction collecting procedures, some means is required to capture cells after separation. The use of solutions that form gels was therefore investigated as a means of maintaining cells and/or macromolecules in the separated state after two types of simple ground-based experiments. Microgravity electrophoresis experiments were simulated by separating model cell types (rat, chicken, human and rabbit erythrocytes) in a vertical density gradient containing low-conductivity buffer, 1.7%-6.5% Ficoll, 6.8-5.0% sucrose, and 1% SeaPrep low-melting temperature agarose and demonstrating that, upon cooling, a gel formed in the column, and cells could be captured in the positions to which they had migrated. Two-phase extraction experiments were simulated by choosing two-polymer solutions in which phase separation occurs in normal saline at temperatures compatible with cell viability and in which one or both phases form a gel upon cooling. Suitable polymers included commercial agaroses (1-2%), maltodextrin (5-7%) and gelatin (5-20%).

Simulation of launch and re-entry acceleration profiles for testing of Shuttle and unmanned microgravity research payloads

Microgravity experiments designed for execution in Get-Away Special canisters, Hitchhiker modules, and Reusable Re-entry Satellites will be subjected to launch and re-entry accelerations. Crew-dependent provisions for preventing acceleration damage to equipment or products will not be available for these payloads during flight; therefore, the effects of launch and re-entry accelerations on all aspects of such payloads must be evaluated prior to flight. A procedure was developed for conveniently simulating the launch and re-entry acceleration profiles of the Space Shuttle (3.3 and 1.7 x g maximum, respectively) and of two versions of NASAs proposed materials research Re-usable Re-entry Satellite (8 x g maximum in one case and 4 x g in the other). By using the 7 m centrifuge of the Gravitational Plant Physiology Laboratory in Philadelphia it was found possible to simulate the time dependence of these 5 different acceleration episodes for payload masses up to 59 kg. A commercial low-cost payload device, the Materials Dispersion Apparatus of Instrumentation Technology Associates was tested for (1) integrity of mechanical function, (2) retention of fluid in its compartments, and (3) integrity of products under simulated re-entry g-loads. In particular, the sharp rise from 1 g to maximum g-loading that occurs during re-entry in various unmanned vehicles was successfully simulated, conditions were established for reliable functioning of the MDA, and crystals of 5 proteins suspended in compartments filled with mother liquor were subjected to this acceleration load.

Further analyses of human kidney cell populations separated on the space shuttle

Cultured human embryonic kidney cells were separated into electrophoretic subpopulations in laboratory experiments and in two separation experiments on the STS-8 (Challenger) Space Shuttle flight using the mid-deck Continuous Flow Electrophoretic Separator (CFES). Populations of cells from each fraction were cultured for the lifetime of the cells, and supernatant medium was withdrawn and replaced at 4-day intervals. Withdrawn medium was frozen at -120 degrees C for subsequent analysis. Enzyme assays, antibodies and gel electrophoresis were used as analytical tools for the detection and quantitation of plasminogen activators in these samples. These assays of frozen culture supernatant fluids confirmed the electrophoretic separation of plasminogen-activator producing cells from non-producing cells, the isolation of cells capable of sustained production, and the separation of cells that produce different plasminogen activators from one another.

High Frequency Access to Low Gravity Experimentation in Organic Crystal Growth from Solutions

Organic crystal growers now have access to a wide array of facilities and hardware on space shuttle missions and other international carriers. Agencies providing such access include governmental organizations (such as the NASA Marshall Space Flight Center and the European Space Agency), educational institutions (e.g., the Birmingham and Huntsville campuses of the University of Alabama, the University of Colorado-Boulder, Kansas State University, Massachusetts Institute of Technology, and the University of California-Riverside), and private companies (such as Instrumentation Technology Associates, Intospace GmbH, and Payload Systems Inc.). These organizations have published descriptions of the hardware they have designed for space flight, including hanging-drop vapor diffusion chambers, membrane-based solute diffusion and solvent-transport cells, sliding-block free diffusion cells, sliding film diffusion cells, batch mixing devices, and submerged crystal growth chambers. In recent years one or more of these facilities has been present on nearly every space shuttle mission, and, in most cases, a high level of automation makes this high frequency of experimentation possible. The total number of individual tests of organic crystallization using this array of devices now numbers into the thousands, making organic crystal growth the most frequently practiced low gravity research procedure in the history of space flight. The services of these facilities have been available for several years to corporate entities wishing to determine the structure of organic molecules having high commercial value. Current hardware can be used to perform any of the widely-utilized (and some of the newly-developed) techniques used for organic crystal growth. These techniques include vapor diffusion (hanging and sessile drop), batch growth (thermal gradient and isothermal), dialysis diffusion, osmotic dewatering, step osmotic dewatering, step dialysis diffusion, liquid-liquid (also known as interfacial or double) diffusion, step diffusion, and boundary-layer diffusion. Every one of these methods has resulted in the growth of diffraction-quality crystals in low gravity. In many cases crystals grown in low gravity have been of higher quality than those grown at 1 g. Some of the methods that have yielded superior crystals (e.g., liquid-liquid diffusion) cannot be practiced successfully at 1 g because of buoyancy-driven motions (convection and sedimentation). The practice of crystallization in low gravity, therefore, widens the variety of techniques available to researchers, and the high frequency of access to this microgravity environment allows researchers to continually improve and broaden their crystal growing capability.