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R A Grant

Publications and source records attributed to R A Grant.

At least 19 recordsLinked to original sources
PubMed1992

Three-dimensional structure of Theiler virus.

Theiler murine encephalomyelitis virus strains are categorized into two groups, a neurovirulent group that rapidly kills the host, and a demyelinating group that causes a generally nonlethal infection of motor neurons followed by a persistent infection of the white matter with demyelinating lesions similar to those found in multiple sclerosis. The three-dimensional structure of the DA strain, a member of the demyelinating group, has been determined at 2.8 A resolution. As in other picornaviruses, the icosahedral capsid is formed by the packing of wedge-shaped eight-stranded antiparallel beta barrels. The surface of Theiler virus has large star-shaped plateaus at the fivefold axes and broad depressions spanning the twofold axes. Several unusual structural features are clustered near one edge of the depression. These include two finger-like loops projecting from the surface (one formed by residues 78-85 of VP1, and the other formed by residues 56-65 of VP3) and a third loop containing three cysteines (residues 87, 89, and 91 of VP3), which appear to be covalently modified. Most of the sequence differences between the demyelinating and neurovirulent groups that could play a role in determining pathogenesis map to the surface of the star-shaped plateau. The distribution of these sequence differences on the surface of the virion is consistent with models in which the differences in the pathogenesis of the two groups of Theiler viruses are the result of differences in immunological or receptor-mediated recognition processes.

Animals↗
PubMed1991

Analysis of symmetry and three-dimensional reconstruction of thin gp32*I crystals.

Thin, multilayered crystals of gp32*I were analyzed by negative stain electron microscopy and image processing. Images of untilted crystals exhibited different projection symmetries and structural motifs. Systematic analysis of these images categorized the projections into four types. Areas producing the type 1 projection were reconstructed in three-dimensions from four tilt series containing 111 images. The three-dimensional data has excellent p121 plane group symmetry and reveals that the gp32*I molecule contains two large domains linked together by a small domain. Computer simulations utilizing projection data suggested that the type 2 and 3 projections arise from superposition of type 1 projections related by a 21 screw axis along the projection axis. The three-dimensional reconstruction was utilized in a final simulation that explained the occurrence of the fourth type of projection. This work provides a firm foundation for future high-resolution analysis of the crystal by electron cryomicroscopy.

Crystallography↗
PubMed1986

Alignment and merging of electron microscope images of frozen hydrated crystals of the T4 DNA helix destabilizing protein gp32*I.

Low dose cryoelectron microscopy has been used to record images and electron diffraction patterns of frozen hydrated crystals of the single-stranded DNA binding protein gp32*I. Fourier transforms from 13 image areas, corresponding to approximately 40,000 unit cells, were aligned by a minimal phase residual search and merged by vector addition in reciprocal space. Phases from the resulting composite transform were combined with amplitudes from electron diffraction patterns to reconstruct the projected mass density of the gp32*I crystal at 8.4 A resolution.

Crystallization↗
PubMed1985

Maximizing gene expression from plasmid vectors containing the lambda PL promoter: strategies for overproducing transcription termination factor rho.

We have constructed two plasmids in which transcription of the rho gene from Escherichia coli K-12 is under the control of the lambda phage PL promoter. In p31-356, the normal rho promoter is deleted, but the remainder of the rho leader region, including the ribosome binding site, is present. In p39-AS, the rho leader is completely absent, and the lambda cII ribosome binding site replaces that of rho. Under noninducing conditions, expression of rho protein from these plasmids is repressed by the lambda cI protein in hosts carrying lambda cryptic prophage. Induction using mitomycin C or nalidixic acid in a cryptic lysogen carrying the cI+ repressor resulted in the overproduction of rho protein to levels of 3%-5% of the total cellular protein with p31-356, and to levels of approximately equal to 40% with p39-AS. The overproduced protein is functionally indistinguishable from the rho protein isolated from the K-12 strain W3110, and it can be obtained from cells harboring p39-AS in yields of up to 25 mg of rho per g of cells. In contrast to chemical induction, heat induction in four cryptic lambda lysogens carrying the thermolabile cI857 repressor failed to yield the same high levels of rho protein (with either plasmid). Our results show that chemical induction of PL-containing plasmid expression vectors can serve as a convenient and useful alternative to the commonly used method of heat induction.

Bacteriophage lambda↗
PubMed1985

Escherichia coli transcription termination factor rho has a two-domain structure in its activated form.

Limited tryptic digestion of Escherichia coli transcription termination factor rho [an RNA-dependent nucleoside triphosphatase (NTPase)] yields predominantly two fragments (f1 and f2) when the protein is bound to both poly(C) and ATP. The apparent molecular masses of the two fragments are 31 kDa for f1 and 15 kDa for f2, adding up to the molecular mass of the intact rho polypeptide chain (46 kDa). Sequence analysis of the amino termini demonstrates that f1 is derived from the amino-terminal portion of rho and that the trypsin cleavage that defines f2 occurs at lysine-283. These results suggest that, in the liganded (activated) form, the native rho protein monomer is organized into two distinct structural domains that are separable by a single proteolytic cleavage. The f1 fragment, purified from NaDodSO4/polyacrylamide gels and renatured, binds poly(C) but the f2 fragment does not; neither regains any ATPase activity. ATP- and polynucleotide-dependent changes in the rate of proteolysis and in the character of the fragments produced suggest that rho undergoes a series of conformational transitions as a consequence of RNA binding, NTP binding and NTP hydrolysis. The rate of loss of rho ATPase activity and of intact rho monomers is slower in the presence of adenosine 5'-[gamma-thio]triphosphate than in the presence of either ATP or ADP, indicating that the hydrolysis of ATP may result in different conformational effects than does the binding of this ligand. These findings are discussed within the context of recent models of rho-dependent transcription termination.

Adenosine Triphosphate↗
PubMed1979

Reconstruction of full-thickness loss skin wounds using skin collagen allografts.

Sheets of allogeneic dermal collagen measuring 20 X 15 mm and prepared by trypsin treatment of full-thickness skin were grafted under skin flaps in rats. After 2 to 5 weeks the protective recipient skin was excised and replaced by split-thickness skin isografts which remained viable on their supportive collagen beds. On average such composite grafts maintained 84 per cent of their original size over 3 to 28 weeks and in contrast with split-thickness skin grafts achieved full-thickness reconstruction of the excised skin.

Animals↗
PubMed1978

Nonreversible loss of platelet aggregability induced by calcium deprivation.

Platelets lose their ability to aggregate when deprived of divalent cations. This usually was studied by incubating human citrated platelet-rich plasma with EDTA or EGTA and then adding enough CaCl2 to combine with the chelating agent. Incubation for 5-7 min at 37 degrees C caused irreversible loss of the platelets' ability to adhere to glass and to aggregate with ADP, epinephrine, A23187, vasopressin, or serotonin or upon rewarming after chilling and markedly reduced aggregation with collagen or thrombin. Control samples incubated with saline, CaEDTA, or CaEGTA were not inhibited. Untreated platelets washed and incubated in solutions treated with resins that remove divalent cations lost their ability to aggregate in 30 min. More than about 0.26 mM Mg2+ partially protected the platelets. Unlike aggregation, ADP-induced shape change, clot retraction caused by thrombin or ADP plus reptilase, and thrombin-induced 14C-serotonin release were not inhibited after incubation. Aggregability was not restored by prolonged incubation with CaCl2, adding normal plasma, or washing the platelets. Its loss was temperature and pH dependent, occurring in 2 min at 43 degrees C but not in 7 min at 30 degrees C, and at pH 7.8 but much less at pH 7.2. The defect was not associated with an increase in platelet cyclic AMP, a decrease in metabolic ATP, or the presence of free ADP.

Calcium↗
PubMed1977

Incorporation of stored cell-free dermal collagen allografts into skin wounds: a short term study.

Allogeneic dermal collagen has been evaluated as an alternative to skin autografts for the permanent replacement of lost or damaged skin in the rat. All non-collagenous structures were removed from full-thickness back skin by treatment with a solution of crystalline trypsin. Such dermal collagen preparations were grafted into skin wounds, examined up to 8 1/2 weeks after operation, and compared with excised wounds and skin isografts and allografts of the same initial size. The dermal collagen grafts, particularly when dressed with a sheet of dermal collagen, become recellularised, revascularised and re-epidermalised. Unlike the skin allografts, there was no evidence of cellular rejection. Whereas contracture of the excised wounds was delayed, but not suppressed, by applying a conventional dressing, collagen grafts maintained from 60 to 100 per cent of their original size. In contrast, equivalent skin isografts shrank to 50-60 per cent and showed persistent epidermal hyperplasia.

Animals↗
PubMed1976

ADP-induced inhibition of von Willebrand factor-mediated platelet agglutination.

Human plasma von Willebrand factor (vWF) plus the antibiotic ristocetin, or bovine or porcine vWF alone, agglutinates platelets in either normal human ethylenediaminetetraacetate (EDTA)-treated citrated platelet-rich plasma (PRP) or citrated PRP from patients with the congenital platelet defect thrombasthenia. The prior addition of 1-10 muM ADP, which causes platelet shape change but not aggregation under these conditions, inhibited vWF-mediated agglutination. Inhibition was prevented by 200 muM ATP. Addition of ADP caused prompt reversal of established vWF-mediated agglutination, which resumed when the ADP was enzymatically removed. EDTA-treated, Formalin-fixed, washed normal platelets also underwent vWF-mediated agglutination. ADP was inhibitory only when added before fixation. Epinephrine (40 muM), prostaglandin E1 (7 muM), or serotonin (2 muM) added before fixation caused slight to moderate inhibition but always less than ADP. Platelets from blood chilled before fixation were fully active. Platelets fixed in freshly prepared PRP did not agglutinate as well as those fixed after incubation of PRP, probably because centrifugation exposes the platelets to ADP. It concluded that ADP causes a reversible decrease in the accessibility of the membrane receptor to vWF.

Adenosine Diphosphate↗