Biomedical subjects

T S Baker

Publications and source records attributed to T S Baker.

At least 73 records · Page 4Linked to original sources

PubMed1994

Three-dimensional structure of vaccinia virus-produced human papillomavirus type 1 capsids.

The capsid proteins of papillomavirus self-assemble to form empty capsids or virus-like particles that appear quite similar to naturally occurring virions by conventional electron microscopy. To characterize such virus-like particles more fully, cryoelectron microscopy and image analysis techniques were used to generate three-dimensional reconstructions of capsids produced by vaccinia virus recombinants (V capsids) that expressed human papillomavirus type 1 L1 protein only or both L1 and L2 proteins. All V capsids had 72 pentameric capsomers arranged on a T = 7 icosahedral lattice. Each particle (approximately 60 nm in diameter) consisted of an approximately 2-nm-thick shell of protein with a radius of 22 nm with capsomers that extend approximately 6 nm from the shell. At a resolution of 3.5 nm, both V capsid structures appear identical to the capsid structure of native human papillomavirus type 1 (T. S. Baker, W. W. Newcomb, N. H. Olson, L. M. Cowsert, C. Olson, and J. C. Brown, Biophys. J. 60:1445-1456, 1991), thus implying that expressed and native capsids are structurally equivalent.

Capsid↗

PubMed1994

Elevated plasma stromelysin levels in arthritis.

OBJECTIVE: To determine whether plasma concentrations of stromelysin-1 and gelatinase A are increased in patients with various forms of arthritis. METHODS: A sensitive and specific sandwich enzyme linked immunosorbent assay (ELISA), which employs a murine monoclonal antibody and a rabbit polyclonal antibody to human stromelysin-1, was used to measure plasma stromelysin-1 in 53 healthy subjects, 113 patients with various forms of arthritis and connective tissue diseases, and 65 patients with cancer. Gelatinase A was also measured in these patients using specific polyclonal and monoclonal antibodies to gelatinase A in an ELISA: RESULTS: The plasma concentration of stromelysin-1 (X +/- SEM) was significantly increased (p < 0.001) in patients with rheumatoid arthritis (RA) (187 +/- 14 ng/ml) and systemic lupus erythematosus (SLE) (258 +/- 35 ng/ml) as compared to both healthy control subjects (50 +/- 4 ng/ml) or patients with cancer (61 +/- 20 ng/ml). Plasma stromelysin-1 was also significantly increased in smaller groups of men with osteoarthritis (OA) and gout. In contrast, plasma concentrations of gelatinase A were not significantly increased in patients with RA, OA or gout. In healthy subjects, the concentration of stromelysin-1 was significantly higher in men than women. No correlation was noted between plasma stromelysin-1 levels and age. CONCLUSION: The detection of elevated plasma levels of stromelysin-1 in patients with RA is consistent with increased stromelysin-1 concentrations in inflamed synovial tissues in this disease. The origin of increased plasma stromelysin-1 in SLE is speculative. Measurement of plasma stromelysin-1 may be useful in the diagnosis and management of patients with various forms of arthritis.

Adult↗

PubMed1993

Structure of a human rhinovirus-bivalently bound antibody complex: implications for viral neutralization and antibody flexibility.

The structure of a neutralizing immunoglobulin (monoclonal antibody mAb17-IA), bound to human rhinovirus 14 (HRV14), has been determined by cryo-electron microscopy and image reconstruction. The antibody bound bivalently across icosahedral twofold axes of the virus, and there were no detectable conformational changes in the capsid. Thus, bivalently bound IgGs do not appear to cause gross deformations in the capsid. Differences between the electron density of the constant domains of the bound Fab fragment and IgG structures suggested that conformational changes occur about elbow axes upon bivalent attachment as was previously predicted. No significant density was observed for the Fc fragment, which adds further evidence for a high degree of mobility about the hinge region.

Antibodies, Viral↗

PubMed1993

Structure of a human rhinovirus complexed with its receptor molecule.

Cryoelectron microscopy has been used to determine the structure of a virus when complexed with its glycoprotein cellular receptor. Human rhinovirus 16 complexed with the two amino-terminal, immunoglobulin-like domains of the intercellular adhesion molecule 1 shows that the intercellular adhesion molecule 1 binds into the 12-A deep "canyon" on the viral surface. This result confirms the prediction that the viral-receptor attachment site lies in a cavity inaccessible to the host's antibodies. The atomic structures of human rhinovirus 14 and CD4, homologous to human rhinovirus 16 and intercellular adhesion molecule 1, showed excellent correspondence with observed density, thus establishing the virus-receptor interactions.

Antigens, CD↗

PubMed1993

Preliminary X-ray data analysis of crystalline cowpea chlorotic mottle virus.

Crystals of cowpea chlorotic mottle virus (CCMV) that diffract X-rays to 3.1 A resolution were grown in a succinate-PEG solution buffered at pH 3.3. The crystals are in space group P2(1)2(1)2(1) with unit cell dimensions of a = 381.26 A, b = 381.26 A, and c = 408.59 A. Four particles occupy the unit cell, placing a single virion in the crystallographic asymmetric unit. Diffraction intensities measured from 196 films collected at the Cornell High Energy Synchrotron Source accounted for 55% of the theoretically possible data to 3.2 A. Unit cell dimensions and rotation function analyses of the X-ray data revealed that the particles were organized in a pseudo-tetragonal relationship with the pseudo-fourfold axis along the crystal c axis. Analysis of electron micrographs of two-dimensional crystals of CCMV revealed a remarkable similarity between these and planes of particles perpendicular to the crystallographic c axis in the three-dimensional crystal.

Crystallization↗

PubMed1993

Use of radial density plots to calibrate image magnification for frozen-hydrated specimens.

Accurate magnification calibration for transmission electron microscopy is best achieved with the use of appropriate standards and an objective calibration technique. We have developed a reliable method for calibrating the magnification of images from frozen-hydrated specimens. Invariant features in radial density plots of a standard are compared with the corresponding features in a "defocused" X-ray model of the same standard. Defocused X-ray models were generated to mimic the conditions of cryo-electron microscopy. The technique is demonstrated with polyoma virus, which was used as an internal standard to calibrate micrographs of bovine papilloma virus type 1 and bacteriophage phi X174. Calibrations of the micrographs were estimated to be accurate to 0.35%-0.5%. Accurate scaling of a three-dimensional structure allows additional calibrations to be made with radial density plots computed from two- or three-dimensional data.

Bacteriophages↗

PubMed1993

Early steps in reovirus infection are associated with dramatic changes in supramolecular structure and protein conformation: analysis of virions and subviral particles by cryoelectron microscopy and image reconstruction.

Three structural forms of type 1 Lang reovirus (virions, intermediate subviral particles [ISVPs], and cores) have been examined by cryoelectron microscopy (cryoEM) and image reconstruction at 27 to 32-A resolution. Analysis of the three-dimensional maps and known biochemical composition allows determination of capsid protein location, globular shape, stoichiometry, quaternary organization, and interactions with adjacent capsid proteins. Comparisons of the virion, ISVP and core structures and examination of difference maps reveal dramatic changes in supra-molecular structure and protein conformation that are related to the early steps of reovirus infection. The intact virion (approximately 850-A diam) is designed for environmental stability in which the dsRNA genome is protected not only by tight sigma 3-mu 1, lambda 2-sigma 3, and lambda 2-mu 1 interactions in the outer capsid but also by a densely packed core shell formed primarily by lambda 1 and sigma 2. The segmented genome appears to be packed in a liquid crystalline fashion at radii < 240 A. Depending on viral growth conditions, virions undergo cleavage by enteric or endosomal/lysosomal proteases, to generate the activated ISVP (approximately 800-A diam). This transition involves the release of an outer capsid layer spanning radii from 360 to 427 A that is formed by 60 tetrameric and 60 hexameric clusters of ellipsoidal subunits of sigma 3. The vertex-associated cell attachment protein, sigma 1, also undergoes a striking change from a poorly visualized, more compact form, to an extended, flexible fiber. This conformational change may maximize interactions of sigma 1 with cell surface receptors. Transcription of viral mRNAs is mediated by the core particle (approximately 600-A diam), generated from the ISVP after penetration and uncoating. The transition from ISVP to core involves release of the 12 sigma 1 fibers and the remaining outer capsid layer formed by 200 trimers of rod-shaped mu 1 subunits that span radii from 306 to 395 A. In the virion and ISVP, flower-shaped pentamers of the lambda 2 protein are centered at the vertices. In the ISVP-to-core transition, domains of the lambda 2 subunits rotate and swing upward and outward to form a turret-like structure extending from radii 305 to 400 A, with a diameter of 184 A, and a central channel 84 A wide. This novel conformational change allows the potential diffusion of substrates for transcription and exit of newly synthesized mRNA segments.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Structure of human rhinovirus complexed with Fab fragments from a neutralizing antibody.

We have determined the structure of a human rhinovirus (HRV)-Fab complex by using cryoelectron microscopy and image reconstruction techniques. This is the first view of an intact human virus complexed with a monoclonal Fab (Fab17-IA) for which both atomic structures are known. The surface area on HRV type 14 (HRV14) in contact with Fab17-IA was approximately 500 A2 (5 nm2), which is much larger than the area that constitutes the NIm-IA epitope (on viral protein VP1) defined by natural escape mutants. From modeling studies and electrostatic potential calculations, charged residues outside the neutralizing immunogenic site IA (NIm-IA) were also predicted to be involved in antibody recognition. These predictions were confirmed by site-specific mutations and analysis of the Fab17-IA-HRV14 complex, along with knowledge of the crystallographic structures of HRV14 and Fab17-IA. The bound Fab17-IA reaches across a surface depression (the canyon) and meets a related Fab at the nearest icosahedral twofold axis. By adjusting the elbow angles of the bound Fab fragments from 162 degrees to 198 degrees, an intact antibody molecule can be easily modeled. This, along with aggregation and binding stoichiometry results, supports the earlier proposal that this antibody binds bivalently to the surface of HRV14 across icosahedral twofold axes. One prediction of this model, that the intact canyon-spanning immunoglobulin G molecule would block attachment of the virus to HeLa cells, was confirmed experimentally.

Antibodies, Monoclonal↗

PubMed1992

Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images.

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components are attached. We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; M(r) = 2.7 x 10(6)) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. Using icosahedral particle reconstruction techniques, we determined its structure to 25 A resolution. Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence. The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of approximately 226 A (frozen-hydrated value), and 12 large openings (approximately 63 A in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (approximately 76-140 A diameter). The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center. The trimers are interconnected by 20 A thick "bridges" on the 2-fold axes. These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule.

Acetyltransferases↗

PubMed1992

Identification of a Fab interaction footprint site on an icosahedral virus by cryoelectron microscopy and X-ray crystallography.

Biological processes frequently require the formation of multi-protein or nucleoprotein complexes. Some of these complexes have been produced in homogeneous form, crystallized, and analysed at high resolution by X-ray crystallography (for example, see refs 1-3). Most, however, are too large or too unstable to crystallize. Individual components of such complexes can often be purified and analysed by crystallography. Here we report how the coordinated application of cryoelectron microscopy, three-dimensional image reconstruction, and X-ray crystallography provides a powerful approach to study large, unstable macromolecular complexes. Three-dimensional reconstructions of native cowpea mosaic virus (CMPV) and a complex of CPMV saturated with a Fab fragment of a monoclonal antibody against the virus have been determined at 23 A resolution from low-irradiation images of unstained, frozen-hydrated samples. Despite the nominal resolution of the complex, the physical footprint of the Fab on the capsid surface and the orientation and position of the Fab have been determined to within a few ångstroms by fitting atomic models of CPMV4 and Fab (Kol)5 to reconstructed density maps.

Antibodies, Monoclonal↗

PubMed1992

Atomic structure of single-stranded DNA bacteriophage phi X174 and its functional implications.

The mechanism of DNA ejection, viral assembly and evolution are related to the structure of bacteriophage phi X174. The F protein forms a T = 1 capsid whose major folding motif is the eight-stranded antiparallel beta barrel found in many other icosahedral viruses. Groups of 5 G proteins form 12 dominating spikes that enclose a hydrophilic channel containing some diffuse electron density. Each G protein is a tight beta barrel with its strands running radially outwards and with a topology similar to that of the F protein. The 12 'pilot' H proteins per virion may be partially located in the putative ion channel. The small, basic J protein is associated with the DNA and is situated in an interior cleft of the F protein. Tentatively, there are three regions of partially ordered DNA structure,

Amino Acid Sequence↗

PubMed1992

Cauliflower mosaic virus: a 420 subunit (T = 7), multilayer structure.

The structures of the Cabb-B and CM1841 strains of cauliflower mosaic virus (CaMV) have been solved to about 3 nm resolution from unstained, frozen-hydrated samples that were examined with low-irradiation cryo-electron microscopy and three-dimensional image reconstruction procedures. CaMV is highly susceptible to distortions. Spherical particles, with a maximum diameter of 53.8 nm, are composed of three concentric layers (I-III) of solvent-excluded density that surround a large, solvent-filled cavity (approximately 27 nm dia). The outermost layer (I) contains 72 capsomeric morphological units, with 12 pentavalent pentamers and 60 hexavalent hexamers for a total of 420 subunits (37-42 kDa each) arranged with T = 7 icosahedral symmetry. CaMV is the first example of a T = 7 virus that obeys the rules of stoichiometry proposed for isometric viruses by Caspar and Klug (1962, Cold Spring Harb. Symp. Quant. Biol. 27, 1-24), although the hexameric capsomers exhibit marked departure from the regular sixfold symmetry expected for a structure in which the capsid protein subunits are quasi-equivalently related. The double-stranded DNA genome is distributed in layers II and III along with a portion of the viral protein. The CaMV reconstructions are consistent with the model based on neutron diffraction studies (Kruse et al., 1987, Virology 159, 166-168) and, together, these structural models are discussed in relation to a replication-assembly model (Hull et al., 1987, J. Cell Sci. (Suppl.) 7, 213-229). Remarkable agreement between the reconstructions of CaMV Cabb-B and CM1841 suggests that other strains of CaMV adopt the same basic structure.

Capsid↗

PubMed1991

Pharmacokinetics and immune response of 131I-chimeric mouse/human B72.3 (human gamma 4) monoclonal antibody in humans.

Chimeric B72.3, composed of the V-regions of murine B72.3 and the constant regions of human immunoglobulin G4 heavy and kappa light chain, was administered as a 131I-labeled conjugate to 12 patients with metastatic colon cancer. Seven of these patients had an antibody response after initial infusion, and the immune response was primarily directed to the murine V-region, although a small proportion of the antibody response was directed to topographical epitopes requiring the presence of both murine V-region and human CH-1 and kappa constant regions (neo-epitopes). The pharmacokinetics included a plasma disappearance curve best fit by a two-compartmental model with an alpha t 1/2 of 18 +/- 7 h and a beta t 1/2 of 224 +/- 66 h. A second infusion of the same dose of 131I-chimeric B72.3 was administered to four of these patients 8 wk after the first infusion. Two patients who had a high antibody response to initial infusion had an anamnestic antibody response, and the infused ch-B72.3 rapidly disappeared from the circulation with associated immune complexes and free 131I in the plasma. One patient with no initial antibody response had no antibody response and identical pharmacokinetics on second infusion. One patient with a modest transient antibody response to initial infusion had no antibody response on second infusion and a modest shortening of plasma circulation. Thus, the human immunoglobulin G4 isotype chimeric B72.3 monoclonal antibody has a plasma half-life 6 to 8 times as long as murine B72.3 and retains considerable immunogenicity in some patients which can adversely affect repetitive infusions.

Animals↗

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

Regulation of the phage phi 29 prohead shape and size by the portal vertex.

Bacteriophage phi 29 of Bacillus subtilis packages its double-stranded DNA into a preformed prohead during morphogenesis. The prohead is composed of the scaffold protein gp7, the capsid protein pg8, the portal protein gp10, and the dispensable head fiber protein gp8.5. Our objective was to elucidate the phi 29 prohead assembly pathway and to define the factors that determine prohead shape and size. The structural genes of the phi 29 prohead were cloned and expressed in Escherichia coli individually or in combination to study form determination. The scaffold protein was purified from E. coli as a soluble monomer. In vivo and in vitro studies showed that the scaffolding protein interacted with both the portal vertex and capsid proteins. When the scaffold protein interacted only with the capsid protein in vivo, particles were formed with variable size and shape. However, in the presence of the portal vertex protein, particles with uniform size and shape were produced in vivo. SDS-PAGE analysis showed that the latter particles contained the proteins of the scaffold, capsid, head fiber, and portal vertex. These results suggest that the scaffolding protein serves as the linkage between the portal vertex and the capsid proteins, and that the portal vertex plays a crucial role in regulating the size and shape of the prohead.

Bacillus subtilis↗

PubMed1991

Structural studies of human alpha 2-macroglobulin: concordance between projected views obtained by negative-stain and cryoelectron microscopy.

Two views of native alpha 2-macroglobulin are revealed by electron microscopy of negatively stained samples; in one view the molecule resembles a padlock and in the other, a pair of lips. Interconversion of the two views upon tilting establishes that these are two different projected views of the same structure. Furthermore, the two views are related by a 45 degrees rotation about their major axis because they interconvert when the specimens are titled +/- 22.5 degrees. Negatively stained molecules on Butvar films present a nearly equal distribution of the two views, whereas in frozen-hydrated samples the molecules almost exclusively are oriented in the lip view. Measurements from both views indicate that the alpha 2-macroglobulin molecule is approximately 200 A long and approximately 140 A wide. Our results suggest that alpha 2-macroglobulin is composed of two protomeric units, each in the shape of a twisted letter S. These units are joined together at their ends to form a complex with point group symmetry 222. The 45 degrees interconversion angle between the lip and padlock views support this arrangement. Average images of unstained and stained lips are quite similar, indicating that the native structure is consistently preserved by the two electron microscopy procedures used in this investigation. This is substantiated by the interconversion between the lip and padlock views that occurs when the molecule is rotated 45 degrees [corrected] about its major twofold axis.

Cryopreservation↗

PubMed1991

Structures of bovine and human papillomaviruses. Analysis by cryoelectron microscopy and three-dimensional image reconstruction.

The structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1) were determined at 2.5 nm resolution by cryoelectron microscopy and three dimensional image reconstruction techniques. As expected, the reconstructions showed that both viruses consist of a T = 7 icosahedral capsid (approximately 60 nm in diameter) which surrounds a nucleohistone core. The capsid morphologies of the two viruses are nearly indistinguishable. Each capsid consists of a shell layer (approximately 2 nm thick) of nearly continuous density from which capsomers project radially to a maximum height of approximately 5.8 nm. The five-coordinate (pentavalent) and six-coordinate (hexavalent) capsomers both exhibit distinct five-fold axial symmetry as was observed for SV40 and polyoma viruses. Thus, both genera (papilloma and polyoma) of the papovavirus family have now been shown to have the characteristic "all-pentamer" capsid construction. BPV-1 and HPV-1 capsomers consist of a thick (8.6 nm diameter) trunk that broadens distally to form a regular five-pointed, star-shaped head, and proximally to create the shell layer where capsomers associate. A cylindrical channel (approximately 2.8 nm diameter) extends along the axis of each capsomer from the interior of the virus to a point approximately half way to the capsomer surface. Computationally sectioned views of individual capsomers displayed at decreasing radii show that each of the five capsomer subunits (in both pentavalent and hexavalent capsomers) makes a pronounced (30 degrees) left-handed twist just above the outer surface of the capsid shell. Similar views of the reconstructions also clarify the morphology of intercapsomer contacts. For example, they show how hexavalent capsomers coordinate six neighboring capsomers despite the fact that they contain only five subunits. The system of intercapsomer contacts is indistinguishable in BPV-1 and HPV-1, but quite different from that reported for polyoma virus capsids assembled in vitro from the major capsid protein, VP1 (D. M. Salunke, D. L. D. Caspar, and R. L. Garcea. 1989. Biophys. J. 56:887-900). Thus, because both polyoma and papilloma viruses have all-pentamer capsids, it appears that intracapsomer subunit-subunit interactions which stabilize pentameric capsomers are better preserved evolutionarily than those involved in capsomer-capsomer contacts.

Animals↗

PubMed1991

Three-dimensional structure of myosin subfragment-1 from electron microscopy of sectioned crystals.

Image analysis of electron micrographs of thin-sectioned myosin subfragment-1 (S1) crystals has been used to determine the structure of the myosin head at approximately 25-A resolution. Previous work established that the unit cell of type I crystals of myosin S1 contains eight molecules arranged with orthorhombic space group symmetry P212121 and provided preliminary information on the size and shape of the myosin head (Winkelmann, D. A., H. Mekeel, and I. Rayment. 1985. J. Mol. Biol. 181:487-501). We have applied a systematic method of data collection by electron microscopy to reconstruct the three-dimensional (3D) structure of the S1 crystal lattice. Electron micrographs of thin sections were recorded at angles of up to 50 degrees by tilting the sections about the two orthogonal unit cell axes in sections cut perpendicular to the three major crystallographic axes. The data from six separate tilt series were merged to form a complete data set for 3D reconstruction. This approach has yielded an electron density map of the unit cell of the S1 crystals of sufficient detail. to delineate the molecular envelope of the myosin head. Myosin S1 has a tadpole-shaped molecular envelope that is very similar in appearance to the pear-shaped myosin heads observed by electron microscopy of rotary-shadowed and negatively stained myosin. The molecule is divided into essentially three morphological domains: a large domain on one end of the molecule corresponding to approximately 60% of the total molecular volume, a smaller central domain of approximately 30% of the volume that is separated from the larger domain by a cleft on one side of the molecule, and the smallest domain corresponding to a thin tail-like region containing approximately 10% of the volume. This molecular organization supports models of force generation by myosin which invoke conformational mobility at interdomain junctions within the head.

Actomyosin↗