Biomedical subjects
Publications and source records attributed to K A Taylor.
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The presence of actin and myosin in non-muscle cells suggests that they may be involved in a wide range of cellular contractile activities. The generally accepted view is that interaction between actin and myosin in these cells and in vertebrate smooth muscle, is regulated by the level of phosphorylation of the 20,000-molecular weight (MW) light chain. In the absence of calcium, this light chain is not phosphorylated and the myosin cannot interact with actin. Calcium activates a specific calmodulin-dependent kinase which phosphorylates the light chain, initiating actin-myosin interaction. Although most studies on the role of phosphorylation have concentration on the regulation of actin-activated myosin Mg-ATPase activity, phosphorylation of the light chain also seems to control the assembly of smooth muscle myosin into filaments. Using purified smooth muscle light chain kinase, we have confirmed this observation. We report here studies of myosins isolated from the two non-muscle sources, thymus cells and platelets. We observed that these myosins are assembled into filaments at physiological ionic strength and Mg-ATP concentrations, only when the 20,000-MW light chain is phosphorylated.
Low temperature transmission electron microscopy can be used to study the structure of biological materials in the hydrated state. Spatial averaging techniques are necessary to overcome the radiation damage problems, and for this reason the techniques described are most applicable to crystalline objects. However, with thin, crystalline biological specimens there are no difficulties with preserving periodicity during the freezing process. Improved specimen preparation methods are described which achieve the production of very thin aqueous films with the hydrated specimen embedded in them. Defocused bright field images of frozen, hydrated protein crystals possess a surprisingly high contrast, presumably due to the difference in density of the protein and the aqueous phase. The techniques described have been used to study the crystal structure of hydrated catalase and the outermost cell wall of Spirillum serpens.
When biological specimens are irradiated by the electron beam in the electron microscope, the specimen structure is damaged as a result of molecular excitation, ionization, and subsequent chemical reactions. The radiation damage that occurs in the normal process of electron microscopy is known to present severe limitations for imaging high resolution detail in biological specimens. The question of radiation damage at low temperatures has therefore been investigated with the view in mind of reducing somewhat the rate at which damage occurs. The radiation damage protection found for small molecule (anhydrous) organic compounds is generally rather limited or even non-existent. However, large molecular, hydrated materials show as much as a 10-fold reduction at low temperature in the rate at which radiation damage occurs, relative to the damage rate at room temperature. In the case of hydrated specimens, therefore, low temperature electron microscopy offers an important advantage as part of the overall effort required in obtaining high resolution images of complex biological structures.
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High-resolution electron diffraction patterns have been obtained from frozen, hydrated catalase crystals to demonstrate the feasibility of using a frozen specimen hydration technique. The use of frozen specimens to maintain the hydration of complex biological structures has certain advantages over previously developed liquid hydration techniques.
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Oblique section reconstruction can produce a 3-D image from electron micrographs of a sectioned crystal when the orientation of the section plane is not aligned with the principal planes of the unit cell. We describe here the reconstruction protocol and the specialized computer software for a Fourier space method that can extract 3-D information from 2-D projection images of oblique sections. The protocol encompasses correction for image defects and image distortions, determination and refinement of reciprocal lattices, calculation of the crystal orientation in 3-D space, extraction of periodic information, alignment in Fourier space, determination and deconvolution of section thickness, and calculation of structure factors of the original crystal. A 3-D map of the unit cell can then be computed by standard crystallographic procedures. Oblique section reconstruction provides an alternative to tomography for 3-D imaging of crystalline objects with large unit cells. The method provides an excellent means of obtaining a 3-D transform using electron microscopy for comparison with X-ray diffraction data from native specimens.
We have adapted a real space refinement protocol originally developed for high-resolution crystallographic analysis for use in fitting atomic models of actin filaments and myosin subfragment 1 (S1) to 3-D images of thin-sectioned, plastic-embedded whole muscle. The rationale for this effort is to obtain a refinement protocol that will optimize the fit of the model to the density obtained by electron microscopy and correct for poor geometry introduced during the manual fitting of a high-resolution atomic model into a lower resolution 3-D image. The starting atomic model consisted of a rigor acto-S1 model obtained by X-ray crystallography and helical reconstruction of electron micrographs. This model was rebuilt to fit 3-D images of rigor insect flight muscle at a resolution of 7 nm obtained by electron tomography and image averaging. Our highly constrained real space refinement resulted in modest improvements in the agreement of model and reconstruction but reduced the number of conflicting atomic contacts by 70% without loss of fit to the 3-D density. The methodology seems to be well suited to the derivation of stereochemically reasonable atomic models that are consistent with experimentally determined 3-D reconstructions computed from electron micrographs.
An image of a thin oblique section through a 3D crystal exhibits superlattice periods much larger than the unit cell dimensions of the crystal. Within a superlattice period the contents of the unit cell of the 3D crystal are sampled at different levels, so that a 2D image of the section contains 3D information about the crystal. The 2D Fourier transform of an electron micrograph of such an oblique section thus exhibits superlattice spots, which provide an estimate of the 3D transform of the original crystal. The strengths of the observed spots are reduced from their true values by convolution with a weighting function that depends on section thickness. A method is described that uses phase relationships among symmetry-related structure factors to determine the section thickness and hence the weighting function. Wiener filter deconvolution of the section thickness is performed, in which the filter level is set by the ratio of diffraction spot intensity to background intensity. From the deconvoluted set of structure factors a 3D map of the unit cell can be computed by a standard crystallographic Fourier program. The approach is illustrated with images of oblique sections through rigor insect flight muscle.
Two-dimensional crystalline sheets of Na, K-ATPase were studied in the vitrified, frozen-hydrated state by electron microscopy and image processing. The technique of correlation averaging was used to determine the projected structure. The projection map shows asymmetry between the pair of "alpha beta" protomers comprising a dimer of Na, K-ATPase molecules. The two protomers differ in overall density as well as in shape. One protomer has an oblong shape, whereas the other with higher density has a head and a hook region. Such an asymmetry has not been reported by other laboratories. This asymmetry may either be due to the coexistence of two different conformations of the enzyme in the dimeric form or due to the simultaneous existence of two molecular species of Na, K-ATPase.
Two-dimensional paracrystalline arrays of F-actin have been formed on positively charged lipid layers composed of phosphatidylcholine (PC) and quaternary ammonium surfactants. These quaternary ammonium surfactants were found to be better promoters of two-dimensional order than PC lipid layers mixed with stearylamine. In addition, the length of the hydrocarbon chain was found to influence the achievement of 2-D order. Lipid layers composed of dilauryl-PC and didodecyldimethylammonium bromide, which are saturated C12 lipids, promoted 2-D crystallization better than mixtures of dipalmitoyl-PC, a saturated C16 lipid, and dioctadecyldimethylammonium bromide, a saturated C18 lipid. Thus, the hydrocarbon chain length, which influences lipid layer fluidity, had a significant effect on paracrystal formation. We suggest that quaternary ammonium surfactants may have advantages in some cases for forming ordered arrays on lipid layers. In addition to investigating the effect of lipid layer composition on paracrystal formation, we found that the injection of G-actin rather than F-actin under a fluid lipid layer into a polymerizing solution produced better ordered paracrystals.
Tomography emerges as a powerful methodology for determining the complex architectures of biological specimens that are better regarded from the structural point of view as singular entities. However, once the structure of a sufficiently large number of singular specimens is solved, quite possibly structural patterns start to emerge. This latter situation is addressed here, where the clustering of a set of 3D reconstructions using a novel quantitative approach is presented. In general terms, we propose a new variant of a self-organizing neural network for the unsupervised classification of 3D reconstructions. The novelty of the algorithm lies in its rigorous mathematical formulation that, starting from a large set of noisy input data, finds a set of "representative" items, organized onto an ordered output map, such that the probability density of this set of representative items resembles at its possible best the probability density of the input data. In this study, we evaluate the feasibility of application of the proposed neural approach to the problem of identifying similar 3D motifs within tomograms of insect flight muscle. Our experimental results prove that this technique is suitable for this type of problem, providing the electron microscopy community with a new tool for exploring large sets of tomogram data to find complex patterns.
Rigor cross-bridges show two conformations paired within each 38.7-nm axial repeat. The two forms may express two stages of the cross-bridge cycle during contraction. Differing numbers of myosin heads per cross-bridge and associated helical changes in the thin filament distinguish the two forms and suggest that both myosin heads usually bind to a single thin filament.
Trypanosomosis is the most economically important disease constraint to livestock productivity in sub-Saharan Africa and has significant negative impact in other parts of the world. Livestock are an integral component of farming systems and thus contribute significantly to food and economic security in developing countries. Current methods of control for trypanosomosis are inadequate to prevent the enormous socioeconomic losses resulting from this disease. A vaccine has been viewed as the most desirable control option. However, the complexity of the parasite's antigenic repertoire made development of a vaccine based on the variable surface glycoprotein coat unlikely. As a result, research is now focused on identifying invariant trypanosome components as potential targets for interrupting infection or infection-mediated disease. Immunosuppression appears to be a nearly universal feature of infection with African trypanosomes and thus may represent an essential element of the host-parasite relationship, possibly by reducing the host's ability to mount a protective immune response. Antibody, T cell and macrophage/monocyte responses of infected cattle are depressed in both trypanosusceptible and trypanotolerant breeds of cattle. This review describes the specific T cell and monocyte/macrophage functions that are altered in trypanosome-infected cattle and compares these disorders with those that have been described in the murine model of trypanosomosis. The identification of parasite factors that induce immunosuppression and the mechanisms that mediate depressed immune responses might suggest novel disease intervention strategies.