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PubMed1994

Dissecting the resolution reaction of lambda integrase using suicide Holliday junction substrates.

A reciprocal strand exchange between two DNA helices generates the crossed-strand intermediate, or Holliday junction, which is common to many pathways of homologous and site-specific recombination. The Int family of recombinases are unique in their ability to both make and resolve Holliday junctions. Previous experiments utilizing 'synthetic' att site Holliday junctions to study the mechanisms associated with the cleavage, transfer and ligation of DNA strands have been confined to studying reciprocal strand exchanges (a pair of temporally overlapping strand cleavages). To circumvent this limitation, we have designed synthetic suicide Holliday junctions that make it possible to monitor individual DNA strand cleavage events. These substrates contain a pre-existing nick in the vicinity of the Int binding site; when Int introduces a second nick into these substrates, the 5'OH nucleophile required for ligation (in either the forward or reverse reaction) is lost by diffusion, thus trapping the covalent protein-DNA intermediate. The results indicate that resolution (involving two partner Ints) is stimulated by additional 'cross-core' Ints as a result of enhanced cleavage rates, and not as a result of enhanced co-ordination of cleavage. Several models for the role of the 'cross-core' Ints during resolution are discussed, as well as the usefulness of these substrates for studying additional aspects of the Holliday junction resolution reaction.

Attachment Sites, Microbiological↗

PubMed2004

Frequency and resolution dependence of the anisotropic impedance estimation in cortical bone using time-resolved scanning acoustic microscopy.

The influences of frequency and spatial resolution on the anisotropic impedance estimation of cortical bone was investigated in the frequency range 25-100 MHz. A set of spherically focused transducers provided a spatial resolution in the range from 150 down to about 20 mum. Four embedded cortical bone samples (two male, two female, two donors aged <30 years, two donors aged >70 years) were cut with different orientations relative to the long axis of the femur (0-90 degrees ). From each section, impedance maps were acquired in the C-scan mode. Histogram evaluations showed a similar angular dependence with a characteristic off-axis maximum of the estimated impedance for all samples and frequencies. The impedance values obtained with the 25-MHz transducer were significantly lower than those obtained with the 50- and 100-MHz transducers. Morphological parameters of the macrostructure, for example, size and distribution of the haversian channels and the resulting porosity, were estimated from the high-resolution acoustic images. These structures appeared to have a significant influence on the measured properties of the bone matrix for the low-frequency and low-aperture measurements.

Adult↗

PubMed2001

High resolution, high-throughput amide deuterium exchange-mass spectrometry (DXMS) determination of protein binding site structure and dynamics: utility in pharmaceutical design.

Mass spectrometry-based peptide amide deuterium exchange techniques have proven to be increasingly powerful tools with which protein structure and function can be studied, and are unparalleled in their ability to probe sub-molecular protein dynamics. Despite this promise, the methodology has remained labor-intensive and time consuming, with substantial limitations in comprehensiveness (the extent to which target protein sequence is covered with measurable peptide fragments) and resolution (the degree to which exchange measurements can be ascribed to particular amides). I have developed and integrated a number of improvements to these methodologies into an automated high throughput, high resolution system termed Deuterium Exchange Mass Spectrometry (DXMS). With DXMS, complete sequence coverage and single-amide (amino acid) resolution are now rapidly accomplished. DXMS is designed to work well with large proteins and when only small amounts of material are available for study. Studies can be performed upon a receptor-ligand pair as they exist on or within a living cell (in vivo) without prior purification, allowing effective in situ study of integral membrane protein receptors. We have ambitious initiatives underway to make DXMS widely available both for basic academic research studies and commercial drug discovery efforts. In this paper I present an overview of DXMS technology and highlight some of the benefits it will provide in drug discovery and basic proteomics research.

Amides↗

PubMed2004

AGBNP: an analytic implicit solvent model suitable for molecular dynamics simulations and high-resolution modeling.

We have developed an implicit solvent effective potential (AGBNP) that is suitable for molecular dynamics simulations and high-resolution modeling. It is based on a novel implementation of the pairwise descreening Generalized Born model for the electrostatic component and a new nonpolar hydration free energy estimator. The nonpolar term consists of an estimator for the solute-solvent van der Waals dispersion energy designed to mimic the continuum solvent solute-solvent van der Waals interaction energy, in addition to a surface area term corresponding to the work of cavity formation. AGBNP makes use of a new parameter-free algorithm to calculate the scaling coefficients used in the pairwise descreening scheme to take into account atomic overlaps. The same algorithm is also used to calculate atomic surface areas. We show that excellent agreement is achieved for the GB self-energies and surface areas in comparison to accurate, but much more expensive, numerical evaluations. The parameter-free approach used in AGBNP and the sensitivity of the AGBNP model with respect to large and small conformational changes makes the model suitable for high-resolution modeling of protein loops and receptor sites as well as high-resolution prediction of the structure and thermodynamics of protein-ligand complexes. We present illustrative results for these kinds of benchmarks. The model is fully analytical with first derivatives and is computationally efficient. It has been incorporated into the IMPACT molecular simulation program.

Algorithms↗

PubMed2005

A scoring function for docking ligands to low-resolution protein structures.

We present a docking method that uses a scoring function for protein-ligand docking that is designed to maximize the docking success rate for low-resolution protein structures. We find that the resulting scoring function parameters are very different depending on whether they were optimized for high- or low-resolution protein structures. We show that this docking method can be successfully applied to predict the ligand-binding site of low-resolution structures. For a set of 25 protein-ligand complexes, in 76% of the cases, more than 50% of ligand-contacting residues are correctly predicted (using receptor crystal structures where the binding site is unspecified). Using decoys of the receptor structures having a 4 A RMSD from the native structure, for the same set of complexes, in 72% of the cases, we obtain at least one correctly predicted ligand-contacting residue. Furthermore, using an 81-protein-ligand set described by Jain, in 76 (93.8%) cases, the algorithm correctly predicts more than 50% of the ligand-contacting residues when native protein structures are used. Using 3 A RMSD from native decoys, in all but two cases (97.5%), the algorithm predicts at least one ligand-binding residue correctly. Finally, compared to the previously published Dolores method, for 298 protein-ligand pairs, the number of cases in which at least half of the specific contacts are correctly predicted is more than four times greater.

Algorithms↗

PubMed1987

Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high-resolution scanning and scanning transmission electron microscopy.

This paper describes the use of sputter coating to prepare detergent-extracted cytoskeletons for observation by scanning (SEM), scanning transmission (STEM), inverted contrast STEM, and transmission (TEM) electron microscopy. Sputtered coats of 1-2 nm of platinum or tungsten provide both an adequate secondary electron signal for SEM and good contrast for STEM and TEM. At the same time, the grain size of the coating is sufficiently fine to be just at (platinum) or below (tungsten) the limit of resolution for SEM and STEM. In TEM, the granular structure of platinum coats is resolved, and platinum decoration artifacts are observed on the surface of structures. The platinum is deposited as small islands with a periodic distribution that may reveal information about the underlying molecular structure. This method produces samples that are similar in appearance to replicas prepared by low-angle rotary shadowing with platinum and carbon. However, the sputter-coating method is easier to use; more widely available to investigators; and compatible with SEM, STEM, and TEM. It may also be combined with immunogold and other labeling methods. While TEM provides the highest resolution images of sputter-coated cytoskeletons, it also damages the specimens owing to heating in the beam. In SEM and STEM cytoskeletons are stable and the resolution is adequate to resolve individual microfilaments. The best single method for visualizing cytoskeletons is inverted contrast STEM, which images both the metal-coated cytoskeletal structures and electron-dense material within the nucleus and cytoplasm as white against a dark background. STEM and TEM were both suitable for visualizing colloidal gold particles in immunolabeled samples.

Cytoskeleton↗

PubMed1989

Application of an ultrahigh-resolution scanning electron microscope (UHS-T1) to biological specimens.

In 1985 we developed an ultrahigh-resolution scanning electron microscope with a resolution of 0.5 nm. It is equipped with a field emission gun and an objective lens with a very short focal length. In this study we report a survey of some different preparation techniques and biological specimens using the new scanning electron microscope. Intracellular structures such as cell organelles were observed surprisingly sharper than those observed by ordinary scanning electron microscopes. However, at magnifications over 250,000 x, platinum particles could be discerned as scattered pebbles on the surface of all structures in coated materials. Using an uncoated but conductively stained specimen, we successfully observed ribosomes on a rough endoplasmic reticulum at a direct magnification of 1 million. In these images some protrusions were recognized on the ribosomes. Ferritin and immunoglobulin G were used as samples of biological macromolecules. These samples were observed without metal coating and conductive staining. The ferritin particles appeared as rounded bodies without any substructure on the surface and immunoglobulin G as complexes of three-unit bodies. In the latter the central body might correspond to the Fc fragment and two side ones to Fab fragments. We assume that ultrahigh-resolution scanning electron microscopy is an effective means for observation of the cell fine structures and biological macromolecules. It will open a new research field in biomedicine.

Animals↗

PubMed1991

Resolution limit of serial sections for 3D reconstruction of tubular cristae in rat liver mitochondria.

Following stereo, high resolution scanning electron microscopy (HRSEM) of various rat tissue mitochondria prepared by mild glutaraldehyde fixation followed by freeze cleavage and extraction of the cytosol (Lea and Hollenberg, 1989a), a new model for the ultrastructure of mitochondrial cristae has been developed (Lea and Hollenberg, 1989b). The cristae mitochondriales, previously considered in most cells to have a shelf like structure, were found in many cell types including hepatocytes by HRSEM to be tubes which spanned the mitochondrial matrix and were continuous with the inner mitochondrial membrane at both ends. Despite computer aided, serial, reconstruction of serial thin sections examined in the transmission electron microscope (TEM), tubular cristae in mitochondria have not been resolved entirely, even though circular cross sectional profiles of cristae have been observed in the TEM. The current study was undertaken to probe this deficiency and to understand its cause. In this study, the problems of inherent contrast and resolution loss in the digitized image have been reduced by using a computer based, background, extraction process analogous in the computer to the chemical cytosol extraction process used for HRSEM (Lea and Hollenberg, 1988; Hollenberg et al., 1989). Despite the use of this membrane enhancement algorithm, it was still not possible to reconstruct entire tubular cristae, as observed by HRSEM, even when the micrograph negatives were digitized and reprocessed (Lea and Hollenberg, 1989b). A comparison of serial section thickness deduced from the diffraction colour of the respective section (60 nm) to the measured diameter of tubular cristae profiles (30 nm), with membrane thickness of 10 nm, suggested that this size disparity is responsible for the apparent loss of resolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

High-resolution scanning electron micrographs of freeze-cracked cells in testes from normal and irradiated rats at different stages of gonadal development.

Newly developed techniques in high-resolution scanning electron microscopy (SEM) and for tissue-processing procedures have been applied to an investigation of structures of various cells in rat testes at different stages of gonadal maturation. A series of high-resolution SEM micrographs are presented which survey the surfaces of different types of testis cells during normal development, and which also illustrate ultrastructural features of some of their intracellular organelles. In addition, a series of high-resolution SEM micrographs are presented which compare the structural features of Sertoli cells in normal testes with those in germ-cell-depleted testes obtained from rats killed at varying times after having been irradiated in utero. We describe our observations on the structural properties of surfaces and intracellular organelles in Sertoli cells, Leydig cells, peritubular myoid cells, and some classes of germinal cells. We also consider the possible role of Sertoli cell apical cytoplasmic processes in lumen formation. Similarities are pointed out between the structure of germ-cell-depleted testes, resulting from irradiation in utero, and the structure of germ-cell-depleted testes in seasonal breeders during periods of involution. Finally, we discuss advantages and disadvantages of methods employed to reveal the fine structure of intracellular organelles in cells of the testis.

Animals↗

PubMed1994

Variations in mitochondrial ultrastructure and dynamics observed by high resolution scanning electron microscopy (HRSEM).

Rat adrenal cortex was processed for high resolution scanning electron microscopy (HRSEM) to confirm tubular cristae, reported by transmission electron microscopy to be present in cortex mitochondria. Mitochondria in several other tissue and cell types were also observed and their ultrastructure confirmed by using three-dimensional, stereo, high resolution scanning electron microscopy. The mitochondria in rat and human hepatocytes as well as human skin fibroblasts grown in culture contained tubular cristae approximately 30 nanometers in diameter. The fibroblast mitochondria proved to be long, up to 46 micrometers and branching, as compared to those in liver which were spherical in shape. Cold adapted brown fat cells were packed with mitochondria, these containing plate or shelf-like cristae. Branched, rat striated muscle mitochondria were observed to curve around contractile protein filament bundles. The muscle mitochondrial cristae were found to be both tubular and plate-like, within the same mitochondrion. The ratio of tubular cristae to plate-like cristae varied considerably between muscle mitochondria. In order to use ultrastructural changes in mitochondria for differential diagnosis, and because 3D reconstruction of mitochondria based on transmission electron microscopy serial sections is severely limited in resolution, it is imperative to first develop a correct understanding of tissue specific, normal mitochondrial ultrastructure based on three-dimensional, HRSEM methods.

Adipose Tissue↗

PubMed2005

Embedding of large specimens in glycol methacrylate: prerequisites for multi-signal detection and high-resolution imaging.

Acrylic resin mixtures are commonly used to study microscopic sections of biological specimens, giving the advantage of good morphological preservation. Existing embedding protocols, however, are suitable for tissue blocks, not exceeding 1 mm in thickness. We have developed a protocol to embed larger specimens (up to 2 cm(3)) in Technovit 8100. This medium allowed us to perform classic histological (trichrome), silver, as well as immunohistochemical staining, needed for multi-signal detection at high-resolution imaging to reconstruct a three-dimensional interpretation of a serially sectioned muscle. The technique was applied to reconstruct the semitendinosus muscle of a fetal pig, 44 days post conception, featuring connective tissue, intramuscular nerves, blood vessels, and muscle fibre types. For the reconstruction, a technique was used that enabled us to insert high-resolution images of histological details into low-resolution images of the entire muscle.

Animals↗

PubMed1990

X-ray crystal structure of the bovine alpha-chymotrypsin/eglin c complex at 2.6 A resolution.

The crystal structure of the molecular complex formed by bovine alpha-chymotrypsin and the recombinant serine proteinase inhibitor eglin c from Hirudo medicinalis has been solved using monoclinic crystals of the complex, reported previously. Four circle diffractometer data at 3.0 A resolution were employed to determine the structure by molecular replacement techniques. Bovine alpha-chymotrypsin alone was used as the search model; it allowed us to correctly orient and translate the enzyme in the unit cell and to obtain sufficient electron density for positioning the eglin c molecule. After independent rigid body refinement of the two complex components, the molecular model yielded a crystallographic R factor of 0.39. Five iterative cycles of restrained crystallographic refinement and model building were conducted, gradually increasing resolution. The current R factor at 2.6 A resolution (diffractometer data) is 0.18. The model includes 56 solvent molecules. Eglin c binds to bovine alpha-chymotrypsin in a manner consistent with other known serine proteinase/inhibitor complex structures. The reactive site loop shows the expected conformation for productive binding and is in tight contact with bovine alpha-chymotrypsin between subsites P3 and P'2; Leu 451 acts as the P1 residue, located in the primary specificity S1 site of the enzyme. Hydrogen bonds equivalent to those observed in complexes of trypsin(ogen) with the pancreatic basic- and secretory-inhibitors are found around the scissile peptide bond.

Animals↗

PubMed2002

Structure of the water resonance in small voxels in rat brain detected with high spectral and spatial resolution MRI.

PURPOSE: To acquire high spectral and spatial resolution (HiSS) MR images of the water resonance in rat brain, evaluate the lineshape of the water resonance in small voxels, and compare images derived from HiSS data with conventional images. MATERIALS AND METHODS: Spectroscopic images of rat brain were obtained at 4.7 Tesla using phase encoding gradients only. Spectral resolution in each voxel was approximately 8 Hz and bandwidth was 1,000 Hz. Spatial resolution was approximately 250 microns in 1-mm slices. Images were synthesized to show the water signal integral, peak height, linewidth, resonance frequency, and asymmetry. RESULTS: Two or more resolved components of the water resonance were detected in approximately 14% +/- 6% of voxels in the brains of eight rats. The water resonances in approximately 20% +/- 10% of voxels (n = 8) were highly asymmetric. Images with intensity proportional to water signal peak height, T(2)*, or to selected components of the water resonance showed features that were not evident in conventional images. CONCLUSIONS: The complexity of the water signal reflects the anatomy and physiology of the sub-voxelar environment, and may be a useful source of image contrast. HiSS imaging of brain provides accurate anatomic information, and may improve image contrast and delineation of subtle anatomic features.

Animals↗

PubMed2002

Combined high-resolution and real-time imaging: a technical feasibility study on coronary magnetic resonance angiography.

PURPOSE: To propose a new approach to combining high-resolution and real-time imaging and to show its technical feasibility on the example of coronary magnetic resonance angiography. MATERIALS AND METHODS: The insertion of fast two-dimensional (2D) acquisitions into time intervals that have not been utilized by triggered or gated 2D or three-dimensional (3D) acquisitions so far is suggested, as well as the immediate reconstruction and display of the additional data. For a technical validation of this concept, a 2D ventricular function protocol was interleaved into a cardiac-triggered and respiratory-gated 3D coronary angiography protocol. Dedicated hardware was employed to rapidly process the data originating from the former. Since the sampling of the latter was restricted to intervals with minimal motion, remaining periods of time could be used to simultaneously image the cardiac and respiratory motion. RESULTS: The technical feasibility of the proposed approach was demonstrated by successful measurements with the combined high-resolution and real-time protocol in volunteers. All examinations provided short axis views during the acquisition and angiograms of selected parts of the coronary system after its completion. CONCLUSION: The investigated concept allows high-resolution measurements to be complemented with real-time imaging functionality without affecting the scan time or image quality. In the particular application considered, an image-based patient monitoring or motion correction is enabled, indicating potential benefits of combining two very dissimilar methods of data acquisition in one measurement.

Coronary Circulation↗

PubMed2003

Fast, high-resolution in vivo cine magnetic resonance imaging in normal and failing mouse hearts on a vertical 11.7 T system.

PURPOSE: To establish fast, high-resolution in vivo cine magnetic resonance imaging (cine-MRI) on a vertical 11.7-T MR system and to investigate the stability of normal and failing mouse hearts in the vertical position. MATERIALS AND METHODS: To optimize the method on a high-field system, various MR-related parameters, such as relaxation times and the need for respiratory gating, were quantitatively investigated. High-resolution cine-MRI was applied to normal mice and to a murine heart failure model. Cardiac functional parameters were compared to matched mice imaged previously on a horizontal MR system. RESULTS: A T(1) of 1.10 +/- 0.27 seconds and a T(2) of 18.5 +/- 3.9 msec were measured for murine myocardial tissue. A quantitative analysis also proved respiratory gating to be essential for obtaining artifact-free cine images in the vertical position at this field strength. Cardiac functional parameters of mice, obtained within one hour, agreed well with those from previous studies of mice in the horizontal position. CONCLUSION: This work shows that MR systems with a vertical bore design can be used to accurately measure cardiac function in both normal and chronically failing mouse hearts within one hour. The increased signal-to-noise ratio (SNR) due to the higher field strength could be exploited to obtain higher temporal and spatial resolution compared to previous studies that were performed on horizontal systems with lower field strengths.

Animals↗

PubMed2001

An investigation into the use of sensitivity-encoded techniques to increase temporal resolution in dynamic contrast-enhanced breast imaging.

Gadolinium-enhanced dynamic magnetic resonance (MR) imaging is playing an increasingly important diagnostic role in patients with breast cancer. Because of the multi-focal nature of the disease, it is mandatory to cover all of both breasts, not only in the initial scan, but also at subsequent follow-up. This requires volume acquisitions with a temporal resolution limited to 60-80 seconds, which is insufficient to clearly discriminate malignant from benign rates of contrast uptake. In this work, we performed sensitivity-encoded imaging using a commercially available four-channel breast coil (MRI Devices Corporation) on a commercial 0.5-T scanner with moderate gradient performance to give increased temporal resolution in these dynamic contrast-enhanced scans. A two-fold increase in temporal resolution was readily achievable with this coil. Image reconstruction was robust and image quality was assessed qualitatively to be good. We also investigated higher speed-up factors using two directions of sensitivity-encoded reduction and discussed some of the potential artifacts associated with such imaging.

Artifacts↗

PubMedDate not supplied

High resolution T2-weighted imaging of the human brain using surface coils and an analytical reception profile correction.

High spatial resolution T2-weighted MR images of the human brain were obtained at 1.5 T. An optimized fast spin-echo (FSE) sequence and 1.5 g/cm gradients were used to obtain T2-weighted images in 4 to 9 minutes with an in-plane resolution of .27 mm and slice thicknesses from 1.5 to 3 mm. Phased arrays of surface coils were used as receivers, providing increased sensitivity but image intensities dependent on the reception profile of the coils. This image nonuniformity was removed by analyzing the data with a theoretical intensity correction algorithm developed in this laboratory. The FSE sequences, the specialized phased arrays of surface coils, and the intensity correction algorithm allowed improved visualization of nerves within the inner auditory canals and surface anatomy of the cerebral cortex. It is expected that this technique will be useful for clinical applications that require high resolution imaging of small, superficial structures of the brain.

Algorithms↗

PubMedDate not supplied

High resolution MRI of MCF7 human breast tumors: complemented use of iron oxide microspheres and Gd-DTPA.

The differential capacity of iron oxide microspheres and of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) to serve as contrast agents that can map the microcirculation of MCF7 human breast cancer implanted in nude mice has been examined by high resolution MRI. Modulation of signal intensity in T2*-weighted, gradient-echo images after iron oxide administration and the temporal signal enhancement after Gd-DTPA administration were monitored and analyzed at a spatial resolution of 98 x 98 x 500 microm and 195 x 390 x 1,000 microm, respectively. The pathophysiologic features revealed in the contrast-enhanced images were analyzed in reference to those obtained from the corresponding high resolution T2-weighted, spin-echo images and from histologic sections stained with hematoxylin and eosin and with an endothelial cell marker. The results showed that iron oxide microspheres can aid in the characterization of gross histopathologic features and in the assessment of the distribution of the microvasculature, whereas Gd-DTPA estimates the permeability of the microvessels to this agent and determines the cellularity (cell volume fraction) in the vicinity of the vessels.

Animals↗