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93 records · Page 6
All sourcesNASA NTRSDOE OSTI
NASA NTRS1999

Novel Approaches to Cellular Transplantation from the US Space Program

Research in the treatment of type I diabetes is entering a new era that takes advantage of our knowledge in an ever increasing variety of scientific disciplines. Some may originate from very diverse sources, one of which is the Space Program at National Aeronautics and Space Administration (NASA). The Space Program contributes to diabetes-related research in several treatment modalities. As an ongoing effort for medical monitoring of personnel involved in space exploration activities NASA and the extramural scientific community investigate strategies for noninvasive estimation of blood glucose levels. Part of the effort in the space protein crystal growth program is high-resolution structural analysis insulin as a means to better understand the interaction with its receptor and with host immune components and as a basis for rational design of a "better" insulin molecule. The Space Program is also developing laser technology for potential early cataract detection as well as a noninvasive analyses for addressing preclinical diabetic retinopathy. Finally, NASA developed an exciting cell culture system that affords some unique advantages in the propagation and maintenance of mammalian cells in vitro. The cell culture system was originally designed to maintain cell suspensions with a minimum of hydrodynamic and mechanical sheer while awaiting launch into microgravity. Currently the commercially available NASA bioreactor (Synthecon, Inc., Houston, TX) is used as a research tool in basic and applied cell biology. In recent years there is continued strong interest in cellular transplantation as treatment for type I diabetes. The advantages are the potential for successful long-term amelioration and a minimum risk for morbidity in the event of rejection of the transplanted cells. The pathway to successful application of this strategy is accompanied by several substantial hurdles: (1) isolation and propagation of a suitable uniform donor cell population; (2) management of host immune rejection; (3) protection from the autoimmune component of the disease; and (4) anatomic placement of the engrafted cells that permits timely response to blood sugar levels as well as effective release and deployment of insulin. Bioreactor technology may provide some critical advances for surmounting some of these scientific hurdles. The NASA bioreactor is a horizontally rotating cylinder that is completely filled with culture medium. Gaseous exchange is maintained by a concentric cylinder of permeable silicon. In slow rotation (15-25 rpm) particles of small mass such as cells and tissue aggregates remain suspended in the rotating body of fluid. This novel approach suspends cells without stirring thus, allowing objects of different size mass to colocate and interact in a very low shear environment. In fact, analysis of the forces acting on individual cells in the rotating bioreactor reveals that the cells are continuously falling through the fluid medium. The conditions in the bioreactor permit assembly of cells into aggregates. three-dimensional tissue growth, synthesis of intercellular matrix,10 differentiation, and some sinusoid formations that may serve as a surrogate vasculature for larger tissue segments.

Pellis, Neal R.↗
NASA NTRS2002

Tetragonal Lysozyme Nucleation and Crystal Growth: The Role of the Solution Phase

Lysozyme, and most particularly the tetragonal form of the protein, has become the default standard protein for use in macromolecule crystal nucleation and growth studies. There is a substantial body of experimental evidence, from this and other laboratories, that strongly suggests this proteins crystal nucleation and growth is by addition of associated species that are preformed by standard reversible concentration-driven self association processes in the bulk solution. The evidence includes high resolution AFM studies of the surface packing and of growth unit size at incorporation, fluorescence resonance energy transfer measurements of intermolecular distances in dilute solution, dialysis kinetics, and modeling of the growth rate data. We have developed a selfassociation model for the proteins crystal nucleation and growth. The model accounts for the obtained crystal symmetry, explains the observed surface structures, and shows the importance of the symmetry obtained by self-association in solution to the process as a whole. Further, it indicates that nucleation and crystal growth are not distinct mechanistically, but identical, with the primary difference being the probability that the particle will continue to grow or dissolve. This model also offers a possible mechanism for fluid flow effects on the growth process and how microgravity may affect it. While a single lysozyme molecule is relatively small (M.W. = 14,400), a structured octamer in the 4(sub 3) helix configuration (the proposed average sized growth unit) would have a M.W. = 115,000 and dimensions of 5.6 x 5.6 x 7.6 nm. Direct AFM measurements of growth unit incorporation indicate that units as wide as 11.2 nm and as long as 11.4 nm commonly attach to the crystal. These measurements were made at approximately saturation conditions, and they reflect the sizes of species that both added or desorbed from the crystal surface. The larger and less isotropic the associated species the more likely that it will be oriented to some degree in a flowing boundary layer, even at the low flow velocities measured about macromolecule crystals. Flow-driven effects resulting in misorientation upon addition to and incorporation into the crystal need only be a small fraction of a percentage to significantly affect the resulting crystal. One Earth, concentration gradient driven flow will maintain a high interfacial concentration, i.e., a high level (essentially that of the bulk solution) of solute association at the interface and higher growth rate. Higher growth rates mean an increased probability that misaligned growth units are trapped by subsequent growth layers before they can be desorbed and try again, or that the desorbing species will be smaller than the adsorbing species. In microgravity the extended diffusive boundary layer will lower the interfacial concentration. This results in a net dissociation of aggregated species that diffuse in from the bulk solution, i.e., smaller associated species, which are more likely able to make multiple attempts to correctly bind, yielding higher quality crystals.

Pusey, Marc L.↗
NASA NTRS1998

Determining the Molecular Growth Mechanisms of Tetragonal Lysozyme Crystals

Studies of the growth of tetragonal lysozyme crystals employing atomic force microscopy (AFM) have shown the advantages of this technique in investigating the growth mechanisms of protein crystals [1]. The resolution of these studies was in the micron range, which revealed surface features such as the occurrence of dislocations and 2D nucleation islands, similar to those found in inorganic systems. They clearly showed that the crystals grew by these surface growth mechanisms. However, the studies also revealed some surprising features, such as bimolecular growth step heights and pronounced growth anisotropies on the (110) face, which could not be explained. In previous studies we employed Periodic Bond Chain (PBC) theory to tetragonal lysozyme crystal growth and found that the crystals were constructed by strongly bonded molecular chains forming helices about the 43 axes [2,3]. The helices were connected to each other with weaker bonds. The growth process was shown to proceed by the formation of these 43 helices, resulting in bimolecular growth steps on the (110) face. It was also shown to explain many other observations on tetragonal lysozyme crystal growth. Although PBC analysis is not a new technique [4], it has not been widely used as the mechanisms predicted from it could not be experimentally verified. In this study the growth process of these crystals was investigated, particularly for the (110) face, employing some newly developed high resolution AFM techniques. These techniques allowed individual lysozyme molecules on the crystal faces to be resolved and predictions from PBC analyses to be tested. The analyses had shown that of the two possible packing arrangements on (110) faces, only one would actually occur. Employing the first of the newly developed techniques, these faces were scanned by high resolution AFM. The resulting images were then compared with the theoretically constructed images for the two possible packing arrangements on the (110) face. The theoretical images were constructed by convolution of the crystal surface shape obtained from crystallographic data with the AFM tip shape. The comparison confirmed the prediction that the molecular packing arrangement of these faces corresponded to that for complete 43 helices. The second AFM technique that was developed was used to follow the growth process by measuring the dimensions of individual growth units on the (110) face. Linescans across a growth step, performed near the saturation limit of the crystals, allowed the growth unit dimensions to be measured. These measurements showed that growth on the (110) face proceeded by the formation of new 43 helices from the addition of at least tetramer units in the [110] direction. In the [001] direction growth proceeded by the addition of various aggregate units corresponding to the 4(sub 3) helices.

Li, Huayu↗