The 1200 patients project: creating a new medical model system for clinical implementation of pharmacogenomics.

The paradigm of individualized drug therapy based on genetics is an ideal that is now potentially possible. However, translation of pharmacogenomics into practice has encountered barriers such as limited availability and the high cost of genetic testing, the delays involved, disagreements about interpretation of results, and even lack of understanding about pharmacogenomics in general. We describe our institutional pharmacogenomics-implementation project, "The 1200 Patients Project," a model designed to overcome these barriers and facilitate the availability of pharmacogenomic information for personalized prescribing.

Atomic force microscopy of arthropod gap junctions.

Atomic force microscopy has been used to characterize gap junctions isolated from the hepatopancreas of Nephrops norvegicus. The major polypeptide of these gap junctions is ductin, a highly conserved 16- to 18-kDa protein. The hydrated gap junctions, imaged in phosphate-buffered saline, appeared as membrane plaques with a thickness of 14 nm, consistent with their being a pair of apposing membranes. The upper membrane was removed by force dissection using an increased imaging force. The thickness of the lower membrane was 6 nm, giving a separation or gap between the two membranes of 2 nm. High-resolution images show fine details of the force-dissected extracellular surfaces, as previously reported for vertebrate and heart gap junctions. In addition high-resolution AFM images show for the first time detailed substructure on the cytoplasmic face of hydrated gap junctions of either vertebrate or invertebrate. The plaques had particles on their exposed and force-dissected faces. These particles were packed in a hexagonal lattice (a = b = 8.9 nm on both faces) and had a diameter of approximately 6.5 nm, with a central, pore-like depression. Fourier maps calculated from the AFM data suggested that each particle was composed of six subunits. These images show a marked similarity to the widely accepted structure of the connexon channel of vertebrate gap junctions.

Imaging real-time neurite outgrowth and cytoskeletal reorganization with an atomic force microscope.

An atomic force microscope was used to image the morphology and structural reorganization of rat NIH/3T3 fibroblasts and PC-12 cells growing in petri dishes. NIH/3T3 fibroblasts had a uniform morphology and an extensive cytoskeletal network. Cell thickness varied from approximately 2-3 microns above the nucleus to approximately 20-30 nm over the distal processes, and cytoskeletal fibers as small as 30 nm wide were observed. Imaging over an extended period of time showed a limited degree of cytoskeletal reorganization. Localized force dissection did not induce significant retraction of cellular processes and immediate cell death. Differentiating PC-12 cells with a neuronal phenotype had a nonuniform morphology, abundant cytoskeletal elements, neuritic processes, and growth cones. The cell thickness varied from approximately 5-8 microns over the nucleus to approximately 100-500 nm over the neuritic processes; growth cones approximately 50-700 nm wide and end structures approximately 30-150 nm wide were visible. Repeated imaging showed reorganization of the growth cone, especially the appearance and disappearance of beadlike features and fibrous organization. Thus an atomic force microscope can be used for high-resolution real-time studies of the dynamic subcellular mechanisms that drive cell behavior.

Dynamic micromechanical properties of cultured rat atrial myocytes measured by atomic force microscopy.

The atomic force microscope (AFM) was used to quantify micromechanical properties (i.e., localized to an area of approximately 0.015 microns 2) of cultured rat atrial myocytes. Quiescent cells in calcium-free solution were quite compressible over the nuclear region, e.g., a force of 3-4 nN produced 180-225 nm cell indentation. Transverse stiffness of quiescent cells increased by approximately 2-fold after an increase in extracellular calcium from 0 to 5 mM and by approximately 16-fold after fixation with Formalin. There was five- to eightfold variation in stiffness of quiescent cells over the cell surface, such that stiffness was lowest over the nuclear region, and it increased toward the cell periphery. These regional variations correlated with the cytoskeletal heterogeneity as revealed by the AFM and fluorescence imaging. Localized contractile activity of beating cells could be monitored in terms of the surface deformation with high transverse spatial (approximately 1-3 nm) and temporal (60-100 microseconds) resolutions. Alterations in cell contractile activity with physiological perturbations and dynamic changes in cell stiffness during a single contraction could be observed. These results demonstrate the feasibility of AFM-based characterization of highly localized cellular micromechanical properties. Relationships among localized cell mechanical behavior and the underlying biochemical and/or structural environment, a crucial aspect in understanding cellular (dys)function, can now be directly examined.

Atomic force microscopy of freeze-fracture replicas of rat atrial tissue.

Atomic force microscopy (AFM) has provided three-dimensional (3-D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well-established technique of transmission electron microscopy (TEM) was performed. Freeze-fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large-scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3-D surface contour) and TEM (2-D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.