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At least 127 records · Page 7

Cell Partition in Two Polymer Aqueous Phases

Partition of biological cells in two phase aqueous polymer systems is recognized as a powerful separation technique which is limited by gravity. The synthesis of new, selective polymer ligand conjugates to be used in affinity partition separations is of interest. The two most commonly used polymers in two phase partitioning are dextran and polyethylene glycol. A thorough review of the chemistry of these polymers was begun, particularly in the area of protein attachment. Preliminary studies indicate the importance in affinity partitioning of minimizing gravity induced randomizing forces in the phase separation process. The PEG-protein conjugates that were prepared appear to be ideally suited for achieving high quality purifications in a microgravity environment. An interesting spin-off of this synthetic work was the observation of catalytic activity for certain of our polymer derivatives.

Harris, J. M.↗

Differential partition of virulent Aeromonas salmonicida and attenuated derivatives possessing specific cell surface alterations in polymer aqueous-phase systems

Two-polymer aqueous-phase systems in which partitioning of biological matter between the phases occurs according to surface properties such as hydrophobicity, charge, and lipid composition are used to compare the surface properties of strains of the fish pathogen Aeromonas salmonicida. The differential ability of strains to produce a surface protein array crucial to their virulence, the A layer, and to produce smooth lipopolysaccharide is found to be important in the partitioning behavior of Aeromonas salmonicida. The presence of the A layer is shown to decrease the surface hydrophilicity of the pathogen, and to increase specifically its surface affinity for fatty acid esters of polyethylene glycol. The method has application to the analysis of surface properties crucial to bacterial virulence, and to the selection of strains and mutants with specific surface characteristics.

Van Alstine, J. M.↗

Protein crystal growth; Proceedings of the First International Conference, Stanford University, CA, August 14-16, 1985

Papers are presented on mechanisms of nucleation and growth of protein crystals, the role of purification in the crystallization of proteins and nucleic acids, and the effect of chemical impurities in polyethylene glycol on macromolecular crystallization. Also considered are growth kinetics of tetragonal lysozyme crystals, thermodynamic and kinetic considerations for crystal growth of complex molecules from solution, protein single-crystal growth under microgravity, and growth of organic crystals in a microgravity environment. Papers are also presented on preliminary investigations of protein crystal growth using the Space Shuttle, convective diffusion in protein crystal growth, and the growth and characterization of membrane protein crystals.

Feigelson, R. S.↗

Physical properties of immiscible polymers

The demixing of immiscible polymers in low gravity is discussed. Applications of knowledge gained in this research will provide a better understanding of the role of phase segregation in determining the properties of polymer blends made from immiscible polymers. Knowledge will also be gained regarding the purification of biological materials by partitioning between the two liquid phases formed by solution of the polymers polyethylene glycol and dextran in water. Testing of new apparatus for space flight, extension of affinity phase partitioning, refinement of polymer chemistry, and demixing of isopycnic polymer phases in a one gravity environment are discussed.

Harris, J. Milton↗

Use of hydrophilic polymer coatings for control of electroosmosis and protein adsorption

The purpose of this project was to examine the utility of polyethylene glycol (PEG) and dextran coatings for control of electroosmosis and protein adsorption; electroosmosis is an important, deleterious process affecting electrophoretic separations, and protein adsorption is a factor which needs to be controlled during protein crystal growth to avoid multiple nucleation sites. Performance of the project required use of X-ray photoelectron spectroscopy to refine previously developed synthetic methods. The results of this spectroscopic examination are reported. Measurements of electroosmotic mobility of charged particles in appropriately coated capillaries reveals that a new, one-step route to coating capillaries gives a surface in which electroosmosis is dramatically reduced. Similarly, both PEG and dextran coatings were shown by protein adsorption measurements to be highly effective at reducing protein adsorption on solid surfaces. These results should have impact on future low-g electrophoretic and protein crystal growth experiments.

Harris, J. Milton↗

Controlled method of reducing electrophoretic mobility of various substances

A method of reducing electrophoretic mobility of macromolecules, particles, cells, and the like is provided. The method comprises interacting the particles or cells with a polymer-linked affinity compound composed of: a hydrophilic neutral polymer such as polyethylene glycol, and an affinity component consisting of a hydrophobic compound such as a fatty acid ester, an immunocompound such as an antibody or active fragment thereof or simular macromolecule, or other ligands. The reduction of electrophoretic mobility achieved is directly proportional to the concentration of the polymer-linked affinity compound employed, and the mobility reduction obtainable is up to 100 percent for particular particles and cells. The present invention is advantageous in that analytical electrophoretic separation can not be achieved for macromolecules, particles, and cells whose native surface charge structure had prevented them from being separated by normal electrophoretic means. Depending on the affinity component utilized, separation can be achieved on the basis of specific/irreversible, specific/reversible, semi-specific/reversible, relatively nonspecific/reversible, or relatively nonspecific/irreversible ligand-substance interactions. The present method is also advantageous in that it can be used in a variety of standard laboratory electrophoresis equipment.

Vanalstine, James M.↗

Phase partitioning in space and on earth

The influence of gravity on the efficiency and quality of the impressive separations achievable by bioparticle partitioning is investigated by demixing polymer phase systems in microgravity. The study involves the neutral polymers dextran and polyethylene glycol, which form a two-phase system in aqueous solution at low concentrations. It is found that demixing in low-gravity occurs primarily by coalescence, whereas on earth the demixing occurs because of density differences between the phases.

Van Alstine, James M.↗

Human serum albumin crystals and method of preparation

Human serum albumin (HSA) crystals are provided in the form of tetragonal plates having the space groups P42(sub 1)2, the crystals being grown to sizes in excess of 0.5 mm in two dimensions and a thickness of 0.1 mm. Growth of the crystals is carried out by a hanging drop method wherein a precipitant solution containing polyethylene glycol (PEG) and a phosphate buffer is mixed with an HSA solution, and a droplet of mixed solution is suspended over a well of precipitant solution. Crystals grow to the desired size in 3 to 7 days. Concentration of reagents, pH and other parameters are controlled within prescribed limits. The resulting crystals exhibit a size and quality such as to allow performance of x ray diffraction studies and enable the conduct of drug binding studies as well as genetic engineering studies.

Carter, Daniel C.↗

Three-dimensional structure of human serum albumin

The three-dimensional structure of human serum albumin has been solved at 6.0 A resolution by the method of multiple isomorphous replacement. Crystals were grown from solutions of polyethylene glycol in the infrequently observed space group P42(1)2 and diffracted X-rays to lattice d-spacings of less than 2.9 A. The electron density maps are of high quality and revealed the structure as a predominantly alpha-helical globin protein in which the course of the polypeptide can be traced. The binding loci of several organic compounds have been determined.

Carter, Daniel C.↗

Radial spreading of viscous-gravity currents with solidifying crust

In the present investigation of solidifying-crust effects on the dynamics and surface morphology of radial viscous-gravity currents, polyethylene glycol inflows into the base of a tank holding a cold sucrose solution are used as analogs. As the radial current advanced away from the inlet, its surface solidified and deformed through a combination of folding anf fracturing. When cooling was sufficiently rapid, solid crust formed and caused the spreading rate to increase; progressively colder experiments revealed a sequence of surface morphologies resembling features of cooling lava flows and lava lakes, including multiarmed rift structures with shear offsets and bulbous lobate forms resembling pillow lavas on the ocean floor.

Fink, Jonathan H.↗

Controlled method of reducing electrophoretic mobility of macromolecules, particles, or cells

A method of reducing electrophoretic mobility of macromolecules, particles, cells, and other substances is provided which comprises interacting in a conventional electrophoretic separating procedure, the substances with a polymer-linked affinity compound comprised of a hydrophilic neutral polymer such as polyethylene glycol bound to a second component such as a hydrophobic compound, an immunocompound such as an antibody or antibody active fragment, or a ligand such as a hormone, drug, antigen, or a hapten. The reduction of electrophoretic mobility achieved is directly proportional to the concentration of the polymer-linked affinity compound employed, and such reduction can comprise up to 100 percent for particular particles and cells. The present invention is advantageous in that electrophoretic separation can now be achieved for substances whose native surface charge structure had prevented them from being separated by normal electrophoretic means. Depending on the affinity component utilized, separation can be achieved on the basis of the specific/irreversible, specific/reversible, semi-specific/reversible, relatively nonspecific/reversible, or relatively nonspecific/irreversible ligand-substance interactions.

Vanalstine, James M.↗

Applications of fibrous substrates containing insolubilized phase change polymers

Incorporation of polyethylene glycols into fibrous substrates produces several improved functional properties when they are insolubilized by crosslinking with a methylolamide resin or by polyacetal formation by their reaction with glyoxal. The range of molecular weights of polyols that may be insolubilized is broad as are the curing conditions (0.25-10 min at 80-200C). Most representative fiber types and blends (natural and synthetic) and all types of fabric constructions (woven, nonwoven and knit) have been modified by incorporation of the bound polyols. The most novel property is the thermal adaptability of the modified substrates to many climatic conditions. This adaptability is due to the high latent heat of the crosslinked polyols that function as phase change materials, the hydrophilic nature of the crosslinked polymer and its enhanced thermal conductivity. Other enhanced properties imparted to fabrics include flex and flat abrasion, antimicrobial activity, reduced static charge, resistance to oily soils, resiliency, wind resistance and reduced lint loss. Applications commercialized in the U.S. and Japan include sportswear and skiwear. Several examples of electric sets of properties useful for specific end uses are given. In addition, other uses are biomedical horticultural, aerospace, indoor insulation, automotive interiors and components and packaging material.

Vigo, Tyrone L.↗

Shapes of Venusian 'pancake' domes imply episodic emplacement and silicic composition

The main evidence available for constraining the composition of the large circular 'pancake' domes on Venus is their gross morphology. Laboratory simulations using polyethylene glycol show that the height to diameter (aspect) ratios of domes of a given total volume depend critically on whether their extrusion was continuous or episodic, with more episodes leading to greater cooling and taller domes. Thus without observations of their emplacement, the compositions of Venusian domes cannot be uniquely constrained by their morphology. However, by considering a population of 51 Venusian domes to represent a sampling of many stages during the growth of domes with comparable histories, and by plotting aspect ratio versus total volume, we find that the shapes of the domes are most consistent with episodic emplacement. On Earth this mode of dome growth is found almost exclusively in lavas of dacite to rhyolite composition, strengthening earlier inferences about the presence of evolved magmas on Venus.

Fink, Jonathan H.↗

Crystals of Serum Albumin for Use in Genetic Engineering and Rational Drug Design

Serum albumin crystal forms have been produced which exhibit superior x-ray diffraction quality. The crystals are produced from both recombinant and wild-type human serum albumin, canine, and baboon serum albumin and allow the performance of drug-binding studies as well as genetic engineering studies. The crystals are grown from solutions of polyethylene glycol or ammonium sulphate within prescribed limits during growth times from one to several weeks and include the following space groups: P2(sub 1), C2, P1.

Carter, Daniel C.↗

Crystallization and Initial X-Ray Diffraction Analysis of Human Pyruvate Dehydrogenase

Human pyruvate dehydrogenase (E1) is a component enzyme of the pyruvate dehydrogenase complex. The enzyme catalyzes the decarboxylation of pyruvate followed by a reductive acetylation of lipoyl groups of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. El is an alpha(sub 2)Beta(sub 2) tetrameric assembly of an approximate molecular mass of 154 kDa. The crystals of this recombinant enzyme have been grown from polyethylene glycol 3350 using vapor diffusion method at 295K. The crystals are characterized as orthorhombic, space group P2(sub 1)2(sub 1)2(sub 1), with cell parameters of a = 64.2, b = 126.9 and c = 190.2 A. Crystals diffracted to a minimum d-spacing of 2.5 A. The asymmetric unit contains one alpha(sub 2)Beta(sub 2) tetrameric El assembly, and self-rotation function analysis showed a pseudo-twofold symmetry relating the two monomers.

Ciszak, Ewa↗

Remobilizing the Interfaces of Thermocapillary Driven Bubbles Retarded by the Adsorption of a Surfactant Impurity on the Bubble Surface

Thermocapillary migration is a method for moving bubbles in space in the absence of buoyancy. A temperature gradient is applied to the continuous phase in which a bubble is situated, and the applied gradient impressed on the bubble surface causes one pole of the drop to be cooler than the opposite pole. As the surface tension is a decreasing function of temperature, the cooler pole pulls at the warmer pole, creating a flow which propels the bubble in the direction of the warmer fluid. A major impediment to the practical use of thermocapillarity to direct the movement of bubbles in space is the fact that surfactant impurities which are unavoidably present in the continuous phase can significantly reduce the migration velocity. A surfactant impurity adsorbed onto the bubble interface is swept to the trailing end of the bubble. When bulk concentrations are low (which is the case with an impurity), diffusion of surfactant to the front end is slow relative to convection, and surfactant collects at the back end of the bubble. Collection at the back lowers the surface tension relative to the front end setting up a reverse tension gradient. For buoyancy driven bubble motions in the absence of a thermocapillarity, the tension gradient opposes the surface flow, and reduces the surface and terminal velocities (the interface becomes more solid-like). When thermocapillary forces are present, the reverse tension gradient set up by the surfactant accumulation reduces the temperature tension gradient, and decreases to near zero the thermocapillary velocity. The objective of our research is to develop a method for enhancing the thermocapillary migration of bubbles which have been retarded by the adsorption onto the bubble surface of a surfactant impurity, Our remobilization theory proposes to use surfactant molecules which kinetically rapidly exchange between the bulk and the surface and are at high bulk concentrations. Because the remobilizing surfactant is present at much higher concentrations than the impurity, it adsorbs to the bubble much faster than the impurity when the bubble is formed, and thereby prevents the impurity from adsorbing onto the surface. In addition the rapid kinetic exchange and high bulk concentration maintain a saturated surface with a uniform surface concentrations. This prevents retarding surface tension gradients and keeps the velocity high. In our first report last year, we detailed experimental results which verified the theory of remobilization in ground based experiments in which the steady velocity of rising bubbles was measured in a continuous phase consisting of a glycerol/water mixture containing a polyethylene glycol surfactant C12E6 (CH3(CH2)11(OCH2CH2)6OH). In our report this year, we detail our efforts to describe theoretically the remobilization observed. We construct a model in which a bubble rises steadily by buoyancy in a continuous (Newtonian) viscous fluid containing surfactant with a uniform far field bulk concentration. We account for the effects of inertia as well as viscosity in the flow in the continuous phase caused by the bubble motion (order one Reynolds number), and we assume that the bubble shape remains spherical (viscous and inertial forces are smaller than capillary forces, i e. small Weber and capillary numbers). The surfactant distribution is calculated by solving the mass transfer equations including convection and diffusion in the bulk, and finite kinetic exchange the bulk and the surface. Convective effects dominate diffusive mass transfer in the bulk of the liquid (high Peclet numbers) except in a thin boundary layer near the surface. A finite volume method is used to numerically solve the hydrodynamic and mass transfer equations on a staggered grid which accounts specifically for the thin boundary layer. We present the results of the nondimensional drag as a function of the bulk concentration of surfactant for different rates of kinetic exchange, from which we develop criteria for the concentration necessary to develop a prescribed degree of remobilization. The criteria compare favorably with the experimental results.

Palaparthi, Ravi↗

Equilibrium Kinetics Studies and Crystallization Aboard the International Space Station (ISS) Using the Protein Crystallization Apparatus for Microgravity (PCAM)

Protein Crystallization Apparatus in Microgravity (PCAM) trays have been used in Shuttle missions to crystallize proteins in a microgravity environment. The crystallization experiments are 'sitting drops' similar to that in Cryschem trays, but the reservoir solution is soaked in a wick. From early 2001, crystallization experiments are conducted on the International Space Station using mission durations of months rather than two weeks on previous shuttle missions. Experiments were set up in April 2001 on Flight 6A to characterize the time crystallization experiments will take to reach equilibrium in a microgravity environment using salts, polyethylene glycols and an organic solvent as precipitants. The experiments were set up to gather data for a series of days of activation with different droplet volumes and precipitants. The experimental set up on ISS and results of this study will be presented. These results will help future users of PCAM to choose precipitants to optimize crystallization conditions for their target macromolecules for a particular mission with known mission duration. Changes in crystal morphology and size between the ground and space grown crystals of a protein and a protein -DNA complex flown on the same mission will also be presented.

Achari, Aniruddha↗

Crystallization and initial X-ray diffraction analysis of human pyruvate dehydrogenase

Human pyruvate dehydrogenase (E1) is a component enzyme of the pyruvate dehydrogenase complex. The enzyme catalyzes the irreversible decarboxylation of pyruvic acid and the rate-limiting reductive acetylation of the lipoyl moiety linked to the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. E1 is an alpha(2)beta(2) tetramer ( approximately 154 kDa). Crystals of this recombinant enzyme have been grown in polyethylene glycol 3350 using a vapor-diffusion method at 295 K. The crystals are characterized as orthorhombic, space group P2(1)2(1)2(1), with unit-cell parameters a = 64.2, b = 126.9, c = 190.2 A. Crystals diffracted to a minimum d spacing of 2.5 A. The asymmetric unit contains one alpha(2)beta(2) tetrameric E1 assembly; self-rotation function analysis showed a pseudo-twofold symmetry relating the two alphabeta dimers.

Pyruvate Dehydrogenase Complex/chemistry↗