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Biomedical subjects

L D Harris

Publications and source records attributed to L D Harris.

At least 19 recordsLinked to original sources
PubMed1989

UvsY protein of bacteriophage T4 is an accessory protein for in vitro catalysis of strand exchange.

The uvsX and uvsY genes are essential to genetic recombination, recombination-dependent DNA synthesis and to the repair of DNA damage in bacteriophage T4. Purified UvsX protein has been shown to catalyze strand exchange and D-loop formation in vitro, but the role of UvsY protein has been unclear. We report that UvsY protein enhances strand exchange by UvsX protein by interacting specifically with UvsX protein: gene 32 protein (gp32) is not necessary for this effect and UvsY protein has no similar effect on the RecA protein of E. coli. UvsY protein, like UvsX protein, protects single-stranded DNA from digestion by nucleases, but, unlike UvsX protein, shows no ability to protect double-stranded DNA. UvsY protein enhances the rate of single-stranded-DNA-dependent ATP hydrolysis by UvsX protein, particularly in the presence of gp32 or high concentrations of salt, factors that otherwise reduce the ATPase activity of UvsX protein. The enhancement of ATP hydrolysis by UvsY protein is shown to result from the ability of UvsY protein to increase the affinity of UvsX protein for single-stranded DNA.

Adenosine Triphosphatases↗
PubMed1988

Formation of D loops by the UvsX protein of T4 bacteriophage: a comparison of the reaction catalyzed in the presence or absence of gene 32 protein.

The UvsX protein of T4 bacteriophage will catalyze the formation of D loops between linear single-stranded DNA (ssDNA) and homologous supercoiled double-stranded DNA (dsDNA) in the absence of T4 gene 32 protein (gp32). This reaction requires one monomer of UvsX protein per three nucleotides of ssDNA so that the ssDNA is completely covered with UvsX protein. Under these conditions, high rates of ATP hydrolysis are observed, and one-third of the products are joined paranemically. The reaction proceeds through a mechanism that creates homology-independent coaggregates of UvsX protein, dsDNA, and ssDNA. When UvsX protein is added to only 1 monomer per 8 nucleotides, but with 1 monomer of gp32 per 12 nucleotides, the rate of ATP hydrolysis is depressed, but D-loop formation is enhanced. Nearly all of the product is bound in plectonemic joints, and no coaggregated intermediates are formed. Coaggregate formation at high concentrations of UvsX protein is not inhibited by the presence of gp32; gp32 simply allows for efficient formation of D loops at such low concentrations of UvsX protein that coaggregates are not constructed. Electron microscopic visualization of the joint structures in this reaction reveals that both gp32 and UvsX protein are bound to the ssDNA. The single-stranded DNA binding (SSB) protein of Escherichia coli will substitute only partially for gp32: in the presence of SSB protein, D-loop formation can be catalyzed at one UvsX protein monomer per eight nucleotides, and it is accomplished without the formation of coaggregates, but a major portion of the product is joined paranemically.

Adenosine Triphosphatases↗
PubMed1988

DNA strand exchanges.

Biochemical and electron microscopic studies of the strand exchange reactions catalyzed by the RecA protein of Escherichia coli and the UvsX protein of T4 phage reveal that these reactions proceed in three distinct steps. The first step, termed joining, involves the assembly of RecA (or UvsX) protein onto a single-stranded DNA (ssDNA) molecule and the subsequent search for homology with a double-stranded DNA (dsDNA) partner and formation of a stable synapsis. In the second step (envelopment/exchange), the exchange of DNA strands occurs fueled by the hydrolysis of ATP. The third step (release of products) entails the resolution of the complexes and dissociation of the protein from the DNAs. The structure of the intermediates in the in vitro reactions catalyzed by the RecA and UvsX proteins is emphasized in this review. The results of pairing different DNA molecules in vitro (such as linear ssDNA pairing with linear or supertwisted dsDNA) are described. Paranemic joints represent a major pathway of joining between two DNA molecules which may involve, in some cases, most of the DNA substrate molecules. Since the nature of paranemic joints has only recently begun to be understood, the nature, role, and possible in vivo function of paranemic joining are considered.

DNA↗
PubMed1987

Visualization of the homologous pairing of DNA catalyzed by the bacteriophage T4 UvsX protein.

The uvsX gene product is essential for DNA repair and general recombination in T4 bacteriophage. The ability of UvsX protein to catalyze the homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA) in vitro was examined by electron microscopic (EM), nitrocellulose filter binding, and gel electrophoretic methods. Optimal joining was observed at ratios of UvsX protein:ssDNA of 2 nucleotides/protein monomer. At this level, the ssDNA was fully covered by UvsX protein as seen by EM, while the dsDNA appeared protein-free. Using this stoichiometry, the pairing of circular ssDNA with homologous supertwisted dsDNA was found to produce a high frequency of complexes in which a supertwisted dsDNA molecule was joined to a UvsX protein-ssDNA filament over a distance of less than 100 base pairs. These joints were labile to deproteinization and must have been paranemic. Pairing of linear ssDNA containing buried homology to the dsDNA produced identical structures. Pairing of fully homologous linear ssDNA and supertwisted dsDNA yielded D-loop joints (plectonemic) as seen by EM following deproteinization. Both the paranemic and the plectonemic joints were at sites of homology, as demonstrated by restriction cleavage of the complexes. Visualization of the joined complexes prior to deproteinization showed that 50% of the joints had the architecture of the paranemic joints, whereas in the remainder, a topologically relaxed dsDNA circle merged with the UvsX protein-ssDNA filament for a distance of 450 base pairs. The structure of the filament was not visibly altered in this region. These observations are similar, but not identical, to findings in parallel studies utilizing the RecA protein of Escherichia coli.

DNA, Bacterial↗
PubMed1985

Three-dimensional cardiac anatomy and function in heart disease in adults: initial results with the dynamic spatial reconstructor.

The dynamic spatial reconstructor, or DSR, is a unique high-speed volume-imaging x-ray scanner based on computed tomographic principles. In this report, we present data obtained from the first feasibility DSR studies of adult patients with heart disease. Information from three patients--one with hypertrophic obstructive cardiomyopathy, one with calcific aortic valvular disease, and one with a left ventricular aneurysm--is described in detail. The mean DSR scanning time for each patient was 20 seconds, and the mean total irradiation to the sternum was 15.3 R. Transverse cross sections were reconstructed and then retrospectively reformatted to provide operator-selected oblique sections in space (for example, long-axis and short-axis sections of the left ventricle), to follow these sections through time (such as from end-diastole through end-systole), and to create three-dimensional displays (for instance, of the left ventricular chamber). Unique quantitative measurements of structure and function were made by using these images. For generation of most imaging data, only one injection of contrast material into the right side of the heart is necessary. Clinically useful three-dimensional dynamic imaging data can be acquired from adult patients with heart disease by using the DSR. Compared with conventional angiocardiography, DSR studies can provide information with less x-ray exposure and fewer angiographic injections.

Adult↗
PubMed1984

The Dynamic Spatial Reconstructor: investigating congenital heart disease in four dimensions.

The Dynamic Spatial Reconstructor (DSR) is a high-temporal resolution, three-dimensional (3-D) X-ray scanning device based on computed tomography (CT) principles. It was designed for investigation of some problems inherent in current diagnostic imaging techniques, and to allow quantitative studies of cardiovascular structure and function. One of the research protocols in which DSR is currently used involves studying selected pediatric patients with complex congenital heart disease. Initial results show that 3-D dynamic images can be obtained from these patients with minimal invasiveness and that these images may provide useful diagnostic information.

Adolescent↗
PubMed1984

Quantitative analysis of a vascular tree model with the dynamic spatial reconstructor.

The accuracy in determining the three-dimensional anatomy of a vessel network by computed tomography (CT) is evaluated using a glass model of a pulmonary artery. The dynamic spatial reconstructor (DSR), a high temporal resolution, volumetric, roentgenographic, CT scanner, was used to scan the model. The glass of the model had a roentgen attenuation coefficient mu = 0.55 cm-1, which is approximately equivalent to the 20% dilution of contrast medium to be expected in the pulmonary arterial tree following a contrast agent bolus injection of 2 ml/kg in the right atrium. The model was scanned inside a 20 cm diameter Plexiglas cylinder with a 1 cm thick wall (mu congruent to 0.2 cm-1) to simulate the chest wall of a 20 kg dog, and it was filled with potato flakes to simulate lung parenchyma (mu congruent to 0.06 cm-1). In one 0.011 s scan, information for reconstruction of a stack of images of transaxial sections was recorded. Sequential scans were performed to obtain data for either maximum transaxial resolution (14 angles of view every 0.0167 s, 120 parallel slices each 1.8 mm thick) or maximum axial resolution (eight angles of view every 0.0167 s, 240 parallel slices each 0.9 mm thick) reconstructions. Estimated detectable "vessel" size, cross-sectional area, branching angle, and interbranch segment length were determined as a function of imaged slice thickness, orientation of section image, and number of angles of view (i.e., scan duration) used to make images. Retrospective selection of 0.05 s duration scan apertures at sequential 0.5 s intervals was used to simulate a typical, retrospectively gated reconstruction from a DSR scan. Using these reconstructed images, 2 mm diameter "vessels" could be readily detected and their structure quantitated. Comparing direct measurements and DSR estimates, cross-sectional area (SEE = 3 mm2), branching angles (SEE = 2 degrees), and segment length (SEE = 1 mm) all had a correlation coefficient greater than 0.99, and the regression lines showed no significant differences from the lines of identity (p greater than 0.05).

Animals↗
PubMed1984

Visualization of SSB-ssDNA complexes active in the assembly of stable RecA-DNA filaments.

We have demonstrated that SSB binds to ssDNA in a complex manner, producing two fundamentally different structures: one that appears as a nucleosomal chain of beads and linkers, and the other as a smooth-contoured, extended nucleoprotein filament. Under physiologic salt conditions, only the beaded complexes were observed. Experiments indicate that the highly beaded forms are the most active in the assembly of the stable RecA-ssDNA filaments. These results further support our previous suggestion (Chrysogelos and Griffith 1982) that the protein-free linker regions are important in two ways. First, they provide access to the DNA template, and second, they provide a means for the two proteins to associate one with the other when bound to ssDNA. We suggest here that SSB should be considered as an assembly factor for RecA in its binding to ssDNA. Furthermore, our results argue that once RecA has associated with both the ssDNA template and SSB, some structural alteration in the (ATP-primed) RecA must occur that nucleates the formation of the very ordered, helical RecA filament along the ssDNA.

DNA, Bacterial↗
PubMed1984

Mass of left ventricular myocardium estimated with dynamic spatial reconstructor.

Using the Dynamic Spatial Reconstructor (DSR), a unique multiple X-ray source, high-repetition-rate CAT scanner, we estimated left ventricular (LV) myocardial volume and chamber volume of eight dogs ranging from 2.5 to 32.5 kg. Dogs were given subcutaneous morphine (3 mg/kg) and anesthetized with intravenous pentobarbital sodium (22 mg/kg). A bolus of 1 ml/kg body wt contrast medium was injected into the superior vena cava and 60/s scans repeated over 7 s were performed. Each 0.0167-s scan generated image data for 120 1.8-mm-thick transverse slices, in the dextro and levo phases of the angiograms. Retrospective reformatting of the scan data was used to generate images of thin slices perpendicular to the aortoapical axis of the LV. The LV muscle and chamber volumes were estimated from their outlines in each imaged slice using a manually operated trackball interfaced to a computer. Values of the LV muscle ranged from 18.0 to 146.8 cm3 by DSR and showed a good correlation with the postmortem values (r = 0.99, y = 0.94x + 4.1). Ratios of volume of the myocardium to chamber volume ranged from 1.19 to 3.10.

Animals↗
PubMed1983

Esophageal manometry in systemic amyloidosis. A study of 30 patients.

The motility of the esophagus was studied by esophageal manometry in 24 patients with primary amyloidosis and six with secondary amyloidosis. Resting lower esophageal sphincter pressure was decreased in 12 patients with primary amyloidosis and two with secondary amyloidosis; 12 of these 14 patients complained of heartburn. Abnormalities in the motility of the body of the esophagus were found in nine patients with primary amyloidosis and one with secondary amyloidosis. No abnormality of the upper esophageal sphincter was demonstrated in any of the 30 patients. Six of the nine patients with primary amyloidosis exhibiting the most marked esophageal motor dysfunction had striking evidence of peripheral and/or autonomic nervous system involvement. No consistent pattern of motility disorder was observed in either group. The manometric abnormalities observed are consistent with a random deposition of amyloid in the esophagus involving a myopathic and/or neuropathic component.

Adult↗
PubMed1982

Dynamic volume imaging of moving organs.

The Dynamic Spatial Reconstructor system has been developed to dynamically (up to 60/sec) image the entire 3-D volume (up to 240 adjacent 1-mm-thick transverse sections) encompassing moving organs of the body, particularly the heart and lungs, or the circulation in any organ. This capability permits accurate regional and global measurements to be made of the important relationships between structure and function within and among these organs, which in turn facilitates achievement of new insights into the basic physiological processes of these organs, and promises increased sensitivity and specificity in the diagnosis of pathology that affects normal organ function. This article explains the biomedical and technological rationale for development of the DSR, describes the design concepts and practical operation of the system, and presents preliminary results obtained with the system, including initial data from one of the first patient studies.

Animals↗
PubMed1982

Three-dimensional spatial, density, and temporal resolution of the dynamic spatial reconstructor.

Spatial, density, and temporal resolution of the dynamic spatial reconstructor (DSR), a multiple X-ray source, high speed, computed tomography scanning system, are evaluated. Hole-pair resolution was evaluated in a stationary phantom surrounded with air, 15 cm of water, or 20 cm of water. Temporal resolution was evaluated by rotation of one of the resolution phantoms during the scan, and with a balloon inflated to a known volume and at a known rate to approximate a typical left ventricular chamber volume and filling rate. These studies confirmed that the spatial resolution is essentially the same in the transverse and axial directions, and that retrospective manipulation of the image data is important for maximization of spatial and density resolution in any structure under examination by obtaining a tradeoff with partial-volume and motion-blurring effects. Maximum spatial resolution in the scanned volume was shown, under ideal conditions, to be greater than five hole pairs per centimeter. Under conditions of intravenous injection of contrast agent, the resolution of blood vessels in an experimental animal approximately 25 kg in weight is expected to be on the order of three hole pairs per centimeter; and in an adult human weighting approximately 60 kg, a resolution of about two hole pairs per centimeter is to be expected.

Angiography↗
PubMed1980

Physics and technical considerations in the design of the DSR: a high temporal resolution volume scanner.

A multiple x-ray source, high-speed, transaxial scanner system (DSR) is about to undergo evaluation studies. The capability for programmable scanning modes and operator-interactive retrospective reconfiguration of scan data makes the DSR a very powerful research tool. The physics and technological basis for system design and selection of several major components of the DSR scanner are discussed.

Humans↗
PubMed1979

Display and visualization of three-dimensional reconstructed anatomic morphology: experience with the thorax, heart, and coronary vasculature of dogs.

A new method, termed reprojection, is used to visualize anatomic morphology contained within three-dimensional reconstructions made up of images of multiple parallel cross sections. This method involves the projection, either orthographically into a plane or radially onto a cylinder, of the volume picture elements (voxels) of the reconstruction. Orthographic reprojection images, formed by mathematically summing the magnitudes of the voxels along selected parallel paths through the reconstructed volume, are analagous to conventional radiographs formed by the passage of an X-ray beam through the volume. The reprojection image is a two-dimensional array of picture elements that is displayed on a television monitor using a digital-to-video scan converter. Also described are the techniques of noninvasive selective tissue dissolution and numerical dissection, whereby obscuring portions of the reconstructed volume are either partially "dissolved" or totally eliminated before reprojection. Utilizing these methods, anatomic information present in a three-dimensional reconstruction but not clearly seen in a reprojection image is rendered visible after removal of superposed structures. The usefulness of these methods is demonstrated utilizing three-dimensional reconstructions of the thorax, heart, and coronary arteries of dogs.

Animals↗
PubMed1979

Distribution of regional volumes and ventilation in excised canine lobes.

A linear elasticity solution for the gravitational deformation of excised lungs was obtained. The accuracy of our solution was examined by comparing predicted and measured displacements of markers glued to the surface of canine lower lobes. The equations describing the strains in a lobe were used to predict the distribution of regional volumes and the slope of phase III (S3) of a single-breath oxygen (SBO2) test. The analysis predicted a negative S3. However, S3 was found to be positive in the five lobes tested, suggesting that factors other than gravity were responsible for the observed pattern of ventilation. In SBO2 tests repeated with increasing delays at end inflation, S3 progressively decreased, became negative, and was eventually abolished. Our equations predicted the most negative observed S3 well. We conclude that continuum mechanics can be used to describe the gravitational deformation of lungs and the resulting effect on ventilation distribution.

Animals↗