Biomedical subjects

B L Trus

Publications and source records attributed to B L Trus.

At least 37 records · Page 2Linked to original sources

PubMed1996

Constructing retinal fundus photomontages. A new computer-based method.

PURPOSE: To develop computer algorithms for reconstructing 24-bit color, wide-angle composite retinal fundus images from a set of adjacent 45 degrees fundus slides. The authors present the description, technical details, and results of the image reconstruction technique. METHODS: Patients with retinal degeneration underwent fundus photography with a 45 degrees field-of-view fundus camera. Individual photographic slides were digitized for creating fundus montages. Background variations in individual 45 degrees images were modeled to first- or second-order two-dimensional polynomial functions to generate a background image. The background image was subtracted from the original image to obtain background corrected image. Background corrected images were registered and spatially transformed using a first- or second-order two-dimensional polynomial warp model to reconstruct a composite retinal fundus montage. RESULTS: The authors successfully reconstructed 24-bit color, 100 degrees field-of-view, composite retinal fundus images. The computer-reconstructed montages are an improvement over manually generated montages because computer analysis can be performed on the computer-based montages. In addition, background variations and discontinuities between individual photographs observed in manually generated montages are reduced greatly in computer-generated montages. Most important, the computer-generated montages are better aligned than the manually generated photomontages. CONCLUSIONS: This method of reconstructing a wide-angle composite retinal fundus image from a set of adjacent small- and wide-angle fundus slides is a new tool for creating montages as large as 100 degrees field of view. The computer-generated montages may be used for documenting and quantifying retinal findings. This can greatly assist studies of retinal manifestations of diseases, such as gyrate atrophy, retinitis pigmentosa, sickle cell disease, and acquired immune deficiency syndrome.

Algorithms↗

PubMed1995

Homology in structural organization between E. coli ClpAP protease and the eukaryotic 26 S proteasome.

Energy-dependent protein degradation is carried out by large multimeric protein complexes such as the proteasomes of eukaryotic and archaeal cells and the ATP-dependent proteases of eubacterial cells. Clp protease, a major multicomponent protease of Escherichia coli, consists of a proteolytic component, ClpP, in association with an ATP-hydrolyzing, chaperonin-like component, ClpA. To provide a structural basis for understanding the regulation and mechanism of action of Clp protease, we have used negative staining electron microscopy and image analysis to examine ClpA and ClpP separately, as well as active ClpAP complexes. Digitized images of ClpP and ClpA were analyzed using a novel algorithm designed to detect rotational symmetries. ClpP is composed of two rings of seven subunits superimposed in bipolar fashion along the axis of rotational symmetry. This structure is similar to that formed by the beta subunits of the eukaryotic and archaeal proteasomes. In the presence of MgATP, ClpA forms an oligomer with 6-fold symmetry when viewed en face. Side views of ClpA indicate that the subunits are bilobed with the respective domains forming two stacked rings. ClpAP complexes contain a tetradecamer of ClpP flanked at one or both ends with a hexamer of ClpA, resulting in a symmetry mismatch between the axially aligned molecules. Our findings demonstrate that, despite the lack of sequence similarity between ClpAP and proteasomes, these multimeric proteases nevertheless have a profound similarity in their underlying architecture that may reflect a common mechanism of action.

Adenosine Triphosphatases↗

PubMed1995

Improved methods for determination of rotational symmetries in macromolecules.

Rotational symmetries of macromolecules are most clearly perceived in the en face projection and may be assessed by inspection of rotational power spectra calculated from electron micrographs of individual particles. However, if the symmetry is not contrasted strongly, this procedure may be inconclusive since the relevant peak may not be convincingly higher than other spectral components. To some extent, this is a sampling problem since the number of repeating elements involved is usually small. We have devised more sensitive statistical tests for rotational symmetry that pool the information contents of entire populations of particles. Both tests involve combining the rotational spectra of many particles and comparing them with the spectra of surrounding background areas. One method is based on the well known t-test which estimates whether two populations differ at a given significance level. In the second test, the ratio between the intensity of each component of the rotational spectrum and the average corresponding intensity for background areas is calculated, and thence, the cumulative product of these ratios over all particles in the data set. If a symmetry is present, this product gradually diverges; otherwise, it converges to zero. As a practical trial, the tests were applied to micrographs of negatively stained hexons of herpes simplex virus and confirmed their 6-fold symmetry. Applied to negatively stained "connector" proteins of bacteriophage T7 purified from a plasmid expression system, both algorithms detected polymorphism with distinct subpopulations of both 13-fold and 12-fold connectors.

Algorithms↗

PubMed1995

Herpes simplex virus capsids assembled in insect cells infected with recombinant baculoviruses: structural authenticity and localization of VP26.

Recently, recombinant baculoviruses have been used to show that expression of six herpes simplex virus type 1 genes results in the formation of capsid-like particles. We have applied cryoelectron microscopy and three-dimensional image reconstruction to establish their structural authenticity to a resolution of approximately 2.7 nm. By comparing capsids assembled with and without the expression of gene UL35, we have confirmed the presence of six copies of its product, VP26 (12 kDa), around each hexon tip. However, VP26 is not present on pentons, indicating that the conformational differences between the hexon and penton states of the major capsid protein, VP5, extend to the VP26 binding site.

Animals↗

PubMed1994

Fungal virus capsids, cytoplasmic compartments for the replication of double-stranded RNA, formed as icosahedral shells of asymmetric Gag dimers.

The primary functions of most virus capsids are to protect the viral genome in the extra-cellular milieu and deliver it to the host. In contrast, the capsids of fungal viruses, like the cores of all other known double stranded RNA viruses, are not involved in host recognition but do shield their genomes, and they also carry out transcription and replication. Nascent (+) strands are extruded from transcribing virions. The capsids of the yeast virus L-A are composed of Gag (capsid protein; 76 kDa), with a few molecules of Gag-Pol (170 kDa). Analysis of these 420 A diameter shells and those of the fungal P4 virus by cryo-electron microscopy and image reconstruction shows that they share the same novel icosahedral structure. Both capsids consist of 60 equivalent Gag dimers, whose two subunits occupy non-equivalent bonding environments. Stoichiometry data on other double-stranded RNA viruses indicate that the 120-subunit structure is widespread, implying that this molecular architecture has features that are particularly favorable to the design of a capsid that is also a biosynthetic compartment.

Capsid↗

PubMed1994

Parallel computing in biomedical research.

Scalable parallel computer architectures provide the computational performance needed for advanced biomedical computing problems. The National Institutes of Health have developed a number of parallel algorithms and techniques useful in determining biological structure and function. These applications include processing electron micrographs to determine the three-dimensional structure of viruses, calculating the solvent-accessible surface area of proteins to help predict the three-dimensional conformation of these molecules from their primary structures, and searching for homologous DNA or amino acid sequences in large biological databases. Timing results demonstrate substantial performance improvements with parallel implementations compared with conventional sequential systems.

Algorithms↗

PubMed1994

Filamentous hemagglutinin of Bordetella pertussis. A bacterial adhesin formed as a 50-nm monomeric rigid rod based on a 19-residue repeat motif rich in beta strands and turns.

The filamentous hemagglutinin (FHA) of Bordetella pertussis is an adhesin that binds the bacteria to cells of the respiratory epithelium in whooping-cough infections. Mature FHA is a 220 kDa secretory protein that is highly immunogenic and has been included in acellular vaccines. We have investigated its structure by combining electron microscopy and circular dichroism spectroscopy (CD) with computational analysis of its amino acid sequence. The FHA molecule is 50 nm in length and has the shape of a horseshoe nail: it has a globular head that appears to consist of two domains; a 35 nm-long shaft that averages 4 nm in width, but tapers slightly from the head end; and a small, flexible, tail. Mass measurements by scanning transmission electron microscopy establish that FHA is a monomer. Its sequence contains two regions of tandem 19-residue pseudo-repeats: the first, of 38 cycles, starts at residue 344; the second, of 13 cycles, starts at residue 1440. The repeat motifs are predicted to consist of short beta-strands separated by beta-turns, and secondary structure measurements by CD support this prediction. We propose a hairpin model for FHA in which the head is composed of the terminal domains; the shaft consists mainly of the repeat regions conformed as amphipathic, hyper-elongated beta-sheets, with their hydrophobic faces apposed; and the tail is composed of the intervening sequence. Further support for the model was obtained by immuno-labeling electron microscopy. The 19-residue repeats of FHA have features in common with the leucine-rich repeats (LRRs) that are present in many eukaryotic proteins, including some adhesion factors. The model is also compared with the two other classes of filamentous proteins that are rich in beta-structure, i.e. viral adhesins and two beta-helical secretory proteins. Our proposed structure implies how the functionally important adhesion sites and epitopes of FHA are distributed: its tripeptide (RGD) integrin-binding site is assigned to the tail; the putative hemagglutination site forms part of the head; and two classes of immunodominant epitopes are assigned to opposite ends of the molecule. Possible mechanisms are discussed for two modes of FHA-mediated adhesion.

Adhesins, Bacterial↗

PubMed1994

Finding a needle in a haystack: detection of a small protein (the 12-kDa VP26) in a large complex (the 200-MDa capsid of herpes simplex virus).

Macromolecular complexes that consist of homopolymeric protein frameworks with additional proteins attached at strategic sites for a variety of structural and functional purposes are widespread in subcellular biology. One such complex is the capsid of herpes simplex virus type 1 whose basic framework consists of 960 copies of the viral protein, VP5 (149 kDa), arranged in an icosahedrally symmetric shell. This shell also contains major amounts of three other proteins, including VP26 (12 kDa), a small protein that is approximately equimolar with VP5 and accounts for approximately 6% of the capsid mass. With a view to inferring the role of VP26 in capsid assembly, we have localized it by quantitative difference imaging based on three-dimensional reconstructions calculated from cryo-electron micrographs. Purified capsids from which VP26 had been removed in vitro by treatment with guanidine hydrochloride were compared with preparations of the same depleted capsids to which purified VP26 had been rebound and with native (undepleted) capsids. The resulting three-dimensional density maps indicate that six VP26 subunits are distributed symmetrically around the outer tip of each hexon protrusion on VP26-containing capsids. Because VP26 may be readily dissociated from and reattached to the capsid, it does not appear to contribute significantly to structural stabilization. Rather, its exposed location suggests that VP26 may be involved in linking the capsid to the surrounding tegument and envelope at a later stage of viral assembly.

Capsid↗

PubMed1993

Structure of the herpes simplex virus capsid. Molecular composition of the pentons and the triplexes.

The molecular anatomy of the herpes simplex virus (HSV-1) capsid has been examined by conventional electron microscopy, cryoelectron microscopy combined with three-dimensional image reconstruction, and scanning transmission electron microscopy (STEM). Studies were carried out with purified capsids before and after treatment with urea and guanidine hydrochloride (GuHCl) at concentrations that maintain the capsid's icosahedral geometry, but selectively extract certain of its protein components. Treatment with 6.0 M urea was found to remove the pentons quantitatively from the capsid vertices, but it caused no appreciable loss of hexons. Penton loss was correlated with solubilization of a small amount of VP5, the major HSV-1 capsid protein, and the amount solubilized (6.1%) was in good agreement with the amount expected (6.3%) if pentons are each composed of five copies of VP5. We conclude that the pentons, like the hexons, are composed of VP5, which exists as a pentamer at the capsid vertices (the pentons) and as a hexamer in all other capsomers (the hexons). Control capsids and capsids extracted with 2.0 M GuHCl (G2.0 capsids) were examined by cryoelectron microscopy and the resulting images were employed to compute three-dimensional reconstructions. Also, the masses of control and G2.0 capsids were determined by dark-field STEM and the results were used to calculate copy numbers for the proteins present. The three-dimensional reconstructions showed that control and G2.0 capsids are similar in structure, except that G2.0 capsids lack all 12 pentons and 120 of the 320 trigonal nodules or "triplexes" that connect HSV-1 capsomers in groups of three. The missing triplexes are the ten closest to each capsid vertex. Thus, the tightness with which triplexes are bound to the VP5 matrix varies according to position on the T = 16 icosahedral surface lattice, those closest to the pentons being most easily detached. Biochemical analyses revealed partial loss of the minor capsid proteins VP19 and VP23 in G2.0 compared to control capsids. Taking into account the STEM data on capsid protein stoichiometry, we propose that HSV-1 triplexes are heterotrimers composed of one copy of VP19 and two copies of VP23.

Capsid↗

PubMed1993

The short tail-fiber of bacteriophage T4: molecular structure and a mechanism for its conformational transition.

Electron microscopy, image processing and computational sequence analysis were used to investigate the structure of the short tail-fiber of bacteriophage T4. This molecule, an oligomer of gp12, is an adhesin that binds the virion irreversibly to the bacterial surface. Short tail-fibers were isolated from mutant-infected cells in which gp12 is synthesized and assembled correctly, but not incorporated into virions. Visualized in negative stain, these filamentous molecules are approximately 38 nm in total length, with an arrowhead-shaped head (approximately 10 nm long by 6 nm wide), a 24-nm shaft of uniform width (approximately 3.8 nm), and a small, seemingly flexible, tail. The primary sequence contains a domain consisting of tandem quasi-repeats, each about 40 residues long, extending from approximately residue 50 to residue 320. Molecular mass analyses by scanning transmission electron microscopy confirm that the molecule is a trimer. The masses of the head, shaft, and tail domains are consistent with (trimers of) the carboxy-terminus, the repeat region, and the amino-terminus, respectively. When short tail-fibers are visualized extending from baseplates, their heads are distal, i.e., detached, implying that it is the tail that remains in contact with the baseplate. Analysis of the molecules' curvature properties detects three hinge-sites: these suggest how the short tail-fiber may be initially accommodated in a compact conformation in the "hexagon" state of the baseplate, from which it converts to the extended conformation when the baseplate switches into its "star" state.

Amino Acid Sequence↗

PubMed1993

Minimum views required to characterize cataracts when using the Scheimpflug camera.

We performed Scheimpflug slit lamp photography and computerized image analysis on 20 normal and 25 cataractous lenses using 18 slit images for each lens taken 10 degrees apart. The data gathered from the normals served as the reference to estimate the accuracy of representation of the cataracts by the least number of views (18 and less) using a Fourier interpolative algorithm. Using the error obtained with one view for the normals, our study suggests that the minimum number of views necessary for adequate characterization is two for cortical cataracts, two for nuclear cataracts, and six for posterior subcapsular cataracts. This information will be useful in longitudinal studies of cataracts, since most researchers presently use only one view, which may be adequate for normals but not for cataractous lenses. We found the Fourier interpolative algorithm useful in estimating the minimum views required for the current method of analyzing Scheimpflug images, and it can be easily applied to other similar images.

Algorithms↗

PubMed1993

Orientation of porin channels in the outer membrane of Bordetella pertussis.

We have examined the surface topography and channel connectivity of a naturally crystalline porin that is known to be functional, and whose structure has not been perturbed by detergent extraction. A three-dimensional density map, calculated from two independent tilt series of negatively stained cell envelopes, reveals three separate channels per trimer on one side (the 'smooth' side), and a single common opening at the other ('rough') side. This arrangement is consistent with the molecular structures recently determined at high resolution by X-ray crystallography for three other porins after detergent solubilization, and implies that the Bordetella pertussis porin may have the same kind of folding. Surface relief maps calculated from electron micrographs of cell envelopes contrasted by unidirectional shadowing clearly show that the side with single opening (i.e. the rough side) represents the external surface.

Bordetella pertussis↗

PubMed1993

Aging studies on normal lens using the Scheimpflug slit-lamp camera.

PURPOSE: To study the changes in density and thickness in normal lenses related to aging, and to study changes in anterior chamber depth related to aging. METHODS: Eighty nine normal volunteers (ages 9-80 yr) were examined and their eyes were photographed to obtain Scheimpflug photographs. The images were digitized and linear densitometry was performed, dividing the lens into five areas: posterior capsular (area 1), posterior cortical (area 2), nuclear (area 3), anterior cortical (area 4), and anterior capsular (area 5). Total lens thickness and anterior chamber depth were similarly measured for 90 normal eyes from the densitometry profiles. These were correlated with age. RESULTS: There was a strong positive correlation between increasing age and the density in all lens areas (area 2: r = 0.805; P < 0.0001; area 3: r = 0.836, P < 0.0001; area 4: r = 0.767, P < 0.0001; and area 5: r = 0.319, P < 0.0023), except the posterior capsular area, where correlation was negative (area 1: r = -0.426; P < 0.0001). In addition, there was a significant correlation between age and overall lens thickness (r = 0.756; P < 0.0001), thickness of nucleus (r = 0.543; P < 0.0001), and cortex (r = 0.632; P < 0.0001), and a negative correlation with anterior chamber depth (r = -0.513, P < 0.0001). CONCLUSION: This report shows human lens changes in density and thickness correlated with aging using Scheimpflug photography and image analysis techniques. The results will aid future development of systems for automated detection, classification, and monitoring of human cataracts, as well as other anterior segment disorders.

Adolescent↗

PubMed1992

Distinct monoclonal antibodies separately label the hexons or the pentons of herpes simplex virus capsid.

The surface shell of the capsid of herpes simplex virus type 1 (HSV-1) is 15 nm thick and 125 nm in outer diameter and has the form of an icosahedral (T = 16) surface lattice, composed of 150 hexons and 12 pentons. Hexons are traversed by axial channels and have six-fold symmetric external protrusions, separated by triangular nodules ("triplexes"). Pentons resemble hexons morphologically, apart from their different order of symmetry. To localize VP5, the major capsid protein, in the shell structure and to investigate whether pentons are composed of the same molecules as hexons, we have performed cryo-electron microscopy and three-dimensional image reconstructions of control HSV-1 B capsids and of B capsids immunoprecipitated with two monoclonal antibodies raised against purified VP5 and purified capsids. The results clearly map the epitope of the anti-VP5 monoclonal antibody to the distal tips of the hexon protrusions. In contrast, no detectable labeling of pentons was observed. We conclude that the hexon protrusions are domains of VP5 hexamers, other parts of these molecules forming the basic matrix of the capsid shell to which the other proteins are attached at specific sites. Conversely, the anti-capsid monoclonal antibody decorates the outer rim of pentons but does not bind to hexons. These observations imply that either pentons are composed of some other protein(s) or that they also contain VP5, but in a conformation sufficiently different from that assumed in hexons as to transform its antigenic character. Other evidence leads us to favor the latter alternative.

Antibodies, Monoclonal↗

PubMed1992

Magnification mismatches between micrographs: corrective procedures and implications for structural analysis.

Quantitative structural analysis from electron micrographs of biological macromolecules inevitably requires the synthesis of data from many parts of the same micrograph and, ultimately, from multiple micrographs. Higher resolutions require the inclusion of progressively more data, and for the particles analyzed to be consistent to within ever more stringent limits. Disparities in magnification between micrographs or even within the field of one micrograph, arising from lens hysteresis or distortions, limit the resolution of such analyses. A quantitative assessment of this effect shows that its severity depends on the size of the particle under study: for particles that are 100 nm in diameter, for example, a 2% discrepancy in magnification restricts the resolution to approximately 5 nm. In this study, we derive and describe the properties of a family of algorithms designed for cross-calibrating the magnifications of particles from different micrographs, or from widely differing parts of the same micrograph. This approach is based on the assumption that all of the particles are of identical size: thus, it is applicable primarily to cryo-electron micrographs in which native dimensions are precisely preserved. As applied to icosahedral virus capsids, this procedure is accurate to within 0.1-0.2%, provided that at least five randomly oriented particles are included in the calculation. The algorithm is stable in the presence of noise levels typical of those encountered in practice, and is readily adaptable to non-isometric particles. It may also be used to discriminate subpopulations of subtly different sizes.

Algorithms↗

PubMed1991

Liquid-crystalline, phage-like packing of encapsidated DNA in herpes simplex virus.

The organization of DNA within the HSV-1 capsid has been determined by cryoelectron microscopy and image reconstruction. Purified C-capsids, which are fully packaged, were compared with A-capsids, which are empty. Unlike A-capsids, C-capsids show fine striations and punctate arrays with a spacing of approximately 2.6 nm. The packaged DNA forms a uniformly dense ball, extending radially as far as the inner surface of the icosahedral (T = 16) capsid shell, whose structure is essentially identical in A-capsids and C-capsids. Thus we find no evidence for the inner T = 4 shell previously reported by Schrag et al. to be present in C-capsids. Encapsidated HSV-1 DNA closely resembles that previously visualized in bacteriophages T4 and lambda, thus supporting the idea of a close parallelism between the respective assembly pathways of a major family of animal viruses (the herpesviruses) and a major family of bacterial viruses.

Animals↗

PubMed1991

Spatial disorders and computational cures.

Image averaging provides a powerful method for enhancing the yield of interpretable information from electron micrographs of biological macromolecules. However, as originally conceived, the full benefit of averaging is achieved only with perfectly ordered two-dimensional crystals. More recent developments, reviewed here, allow one to rectify disordered lattices, straighten randomly bent filaments, and combine multiple images of free-standing particles, thus extending the advantages of image averaging to virtually every class of macromolecular specimen.

Animals↗

PubMed1991

Image averaging of flexible fibrous macromolecules: the clathrin triskelion has an elastic proximal segment.

We have developed computational techniques that allow image averaging to be applied to electron micrographs of filamentous molecules that exhibit tight and variable curvature. These techniques, which involve straightening by cubic-spline interpolation, image classification, and statistical analysis of the molecules' curvature properties, have been applied to purified brain clathrin. This trimeric filamentous protein polymerizes, both in vivo and in vitro, into a wide range of polyhedral structures. Contrasted by low-angle rotary shadowing, dissociated clathrin molecules appear as distinctive three-legged structures, called "triskelions" (E. Ungewickell and D. Branton (1981) Nature 289, 420). We find triskelion legs to vary from 35 to 62 nm in total length, according to an approximately bell-shaped distribution (mu = 51.6 nm). Peaks in averaged curvature profiles mark hinges or sites of enhanced flexibility. Such profiles, calculated for each length class, show that triskelion legs are flexible over their entire lengths. However, three curvature peaks are observed in every case: their locations define a proximal segment of systematically increasing length (14.0-19.0 nm), a mid-segment of fixed length (approximately 12 nm), and a rather variable end-segment (11.6-19.5 nm), terminating in a hinge just before the globular terminal domain (approximately 7.3 nm diameter). Thus, two major factors contribute to the overall variability in leg length: (1) stretching of the proximal segment and (2) stretching of the end-segment and/or scrolling of the terminal domain. The observed elasticity of the proximal segment may reflect phosphorylation of the clathrin light chains.

Animals↗