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Biomedical subjects

D Stuart

Publications and source records attributed to D Stuart.

At least 55 records · Page 3Linked to original sources
PubMed1995

Analysis of antigenic surfaces of proteins.

The processes underlying molecular evolution have proved difficult to understand due to the complexity and obscurity of the selective pressures at work. The strong selective pressure to optimize antigen recognition means that antibody paratopes are more favorable systems than most in which to investigate these processes, as are viral epitopes, which evolve under an inverse pressure, rapidly changing to avoid recognition. Because recognition is a property of the surfaces of these molecules we expect that their evolutionary development may be read in the changes in the 3-dimensional array of chemical groups displayed on their surface. We have analyzed the bulk properties of these surfaces and find that there are significant differences in exposed amino acid preferences among 1) a control group of immunologically secluded proteins, 2) binding surfaces of immunoglobulins, and 3) the outer surfaces of picornaviruses. Compared to the control group, the immunoglobulin complementarity determining regions possess a relative excess of serine residues whereas picornaviruses suppress serine but overuse threonine residues, suggesting that the differing selective pressure has led to perturbations in the population of amino acid types on the surface of these proteins. Although these changes may be rationalized in terms of the structure and chemistry of the different side chains, we suggest that there may be a further, genetic component behind the observations: point mutations in the respective codons for serine and threonine lead to markedly different forms of structural variability. The high rate of reselection observed for these residues is in line with this second mechanism.

Amino Acids↗
PubMed1995

Quantification of the hemolysis associated with use of T-61 as a euthanasia agent in rabbits--a comparison with Euthanyl (pentobarbital sodium) and the impact on serum hexosaminidase measurements.

Albino rabbits (2-2.3 kg) were euthanized with T-61R (N-[2-(m-methoxyphenyl)-2-ethylbutyl-(1)-]-gamma-hydroxybutyram ide, 200 mg/mL; 4,4'-methylene-bis-(cyclohexyltrimethylammonium iodide), 50 mg/mL; tetracaine hydrochloride, 5 mg/mL; 0.2-1.0 mL/kg, i.v.) or EuthanylR (pentobarbital sodium, 100-240 mg/kg, i.v.) at 15:00 h, and samples of arteriovenous blood or venous blood were collected. The time to loss of consciousness was dose dependent for T-61, ranging from 7 to 1 s or less. Serum obtained after T-61 euthanasia, but not after Euthanyl, was routinely contaminated with measurable hemoglobin levels (up to an estimated 3 mg/mL). The extent of hemolysis increased with increasing T-61 doses. The activity of hexosaminidase in the serum (determined by hydrolysis of a methylumbelliferyl substrate at pH 4.6) was up to 80% lower in sera obtained after the use of T-61 compared with sera obtained after the use of Euthanyl or without euthanasia drugs. The reduction in measurable activity appears to be due to interference, by hemoglobin, in the fluorescence measurements of the methylumbelliferol reaction product (with threshold effects being detected at 0.1 mg/mL). Standardization of serum volumes used in these types of enzyme assays and reporting on hemoglobin levels is thus recommended.

Amides↗
PubMed1994

Crystallization and preliminary X-ray investigation of recombinant simian immunodeficiency virus matrix protein.

Simian immunodeficiency virus matrix protein has been crystallized from Tris-HCl buffer with polyethylene glycol and isopropanol as precipitants. The crystals belong to space group C2 with unit cell dimensions a = 69.9 A, b = 115.2 A, c = 32.5 A, beta = 108.1 degrees and diffract X rays to a minimum Bragg spacing of 2 A. These crystals appear suitable for a high resolution structure analysis.

Crystallization↗
PubMed1994

Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2.

The three-dimensional structure of the complex between the Fab fragment of an anti-human rhinovirus neutralizing antibody (8F5) and a cross-reactive synthetic peptide from the viral capsid protein VP2 has been determined at 2.5 A resolution by crystallographic methods. The refinement is presently at an R factor of 0.18 and the antigen-binding site and viral peptide are well defined. The peptide antigen adopts a compact fold by two tight turns and interacts through hydrogen bonds, some with ionic character, and van der Waals contacts with antibody residues from the six hypervariable loops as well as several framework amino acids. The conformation adopted by the peptide is closely related to the corresponding region of the viral protein VP2 on the surface of human rhinovirus 1A whose three-dimensional structure is known. Implications for the cross-reactivity between peptides and the viral capsid are discussed. The peptide-antibody interactions, together with the analysis of mutant viruses that escape neutralization by 8F5 suggest two different mechanisms for viral escape. The comparison between the complexed and uncomplexed antibody structures shows important conformational rearrangements, especially in the hypervariable loops of the heavy chain. Thus, it constitutes a clear example of the 'induced fit' molecular recognition mechanism.

Amino Acid Sequence↗
PubMed1994

Antigenic heterogeneity of a foot-and-mouth disease virus serotype in the field is mediated by very limited sequence variation at several antigenic sites.

Antigenic variation in a major discontinuous site (site D) of foot-and-mouth disease virus (FMDV) of serotype C has been evaluated with neutralizing monoclonal antibodies. Isolates representing the major evolutionary sublines previously defined for serotype C were compared. Extensive variation, comparable to that of continuous epitopes within the hypervariable immunodominant site A (the VP1 G-H loop), was found. The amino acid sequences of the complete capsids of three antigenically highly divergent FMDVs (C1 Haute Loire-Fr/69, C5 Argentina/69, and C3 Argentina/85) have been determined and compared with the corresponding sequences previously determined for seven additional type C viruses. Differences in antigenicity are due to a very limited number of substitutions of surface amino acids accessible to antibodies and located within antigenic sites previously identified on FMDV. A significant number of residues at these positions were also replaced in monoclonal antibody escape mutants. Depending on the variants compared, replacements within site A or at site D, or at both sites, contributed significantly to their antigenic differences. Examples of divergence mediated by a few amino acid replacements were found among FMDVs of Europe and South America. The results suggest that within a serotype of FMDV, antigenically highly divergent viruses can arise in the field by very limited sequence variation at exposed key residues of each of several antigenic sites.

Amino Acid Sequence↗
PubMed1994

Cell cycle-dependent transcription of CLN2 is conferred by multiple distinct cis-acting regulatory elements.

The budding yeast Saccharomyces cerevisiae CLN1, CLN2, and CLN3 genes encode functionally redundant G1 cyclins required for cell cycle initiation. CLN1 and CLN2 mRNAs accumulate periodically throughout the cell cycle, peaking in late G1. We show that cell cycle-dependent fluctuation in CLN2 mRNA is regulated at the level of transcriptional initiation. Mutational analysis of the CLN2 promoter revealed that the major cell cycle-dependent upstream activating sequence (UAS) resides within a 100-bp fragment. This UAS contains three putative SWI4-dependent cell cycle boxes (SCBs) and two putative MluI cell cycle boxes (MCBs). Mutational inactivation of these elements substantially decreased CLN2 promoter activity but failed to eliminate periodic transcription. Similarly, inactivation of SWI4 decreased CLN2 transcription without affecting its periodicity. We have identified a second UAS in the CLN2 upstream region that can promote cell cycle-dependent transcription with kinetics similar to that of the intact CLN2 promoter. Unlike the major CLN2 UAS, this newly identified UAS promotes transcription in cells arrested in G1 by inactivation of cdc28. This novel UAS is both necessary and sufficient for regulated transcription driven by a CLN2 promoter lacking functional SCBs and MCBs. Although this UAS itself contains no SCBs or MCBs, its activity is dependent upon SWI4 function. The characteristics of this novel UAS suggest that it might have a role in initiating CLN2 expression early in G1 to activate the positive feedback loop that drives maximal Cln accumulation.

Base Sequence↗
PubMed1994

Quality down under: if we knew then what we know now.

Australian health care's approach to performance improvement increasingly includes the use of total quality management (TQM) and its component principles and methods. Implementation experiences have confirmed TQM's relevance in the Australian health care environment, senior management's pivotal role, and the range of factors required for success. Ways of influencing executive behavioral change and streamlining implementation are discussed.

Australia↗
PubMed1994

Total quality management in health care.

Studying 'the best way of doing things' does expedite performance improvement. Using cost as the only measure of health care has provided a one dimensional view of 'the best'. Now there is an urgency to improve the value of clinical outcomes, the real product of health care. For this to be achieved by any method, more effective and comparable measures of quality are needed to focus improvement efforts. This paper comments on the application of the best practice principles, developed for other Australian industries, to health care.

Australia↗
PubMed1993

Crystallization and X-ray diffraction study of recombinant platelet-derived endothelial cell growth factor.

Crystals of recombinant platelet-derived endothelial cell growth factor (PD-ECGF) were obtained by the hanging drop vapour diffusion technique. The crystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions a = 63.7 A, b = 70.4 A, c = 219.6, alpha = beta = gamma = 90 degrees, and probably contain a single dimer in the asymmetric unit. Diffraction to a minimum Bragg spacing of 3.5 A has been obtained using a synchrotron X-ray source.

Blood Platelets↗
PubMed1993

Preliminary crystallographic analysis of bovine enterovirus.

Bovine enterovirus (BEV) strain VG-5-27 derived from an infectious cDNA clone has been crystallized as extended hexagonal plates. Virus recovered from crystals produced cytopathic effect in BHK cells. These crystals diffract X-rays from high energy synchrotron sources to beyond 2.7 A. The crystal system is monoclinic, space group P2(1) with unit cell dimensions a = 388 A, b = 390 A, c = 360 A, beta = 113 degrees. The virion is 300 A in diameter and one whole particle constitutes the crystallographic asymmetric unit, giving rise to 60-fold non-crystallographic redundancy.

Animals↗
PubMed1993

Preliminary crystallographic analysis of coxsackievirus A9.

Coxsackievirus A9 has been crystallized as small rhombic dodecahedra of maximum dimension 0.3 mm. These crystals have been shown, using synchrotron radiation, to diffract X-rays to beyond 3 A, and to have a stability in the beam comparable to that of other related virus crystals. The unit cell is tetragonal with dimensions a = b = 495 A, c = 695 A and alpha = beta = gamma = 90 degrees, with a space group of P4n22. A substantial body of diffraction data has been collected and this crystal form appears to be suitable for structure determination. Phasing of these data will be attempted using molecular replacement.

Animals↗
PubMed1993

Methods used in the structure determination of foot-and-mouth disease virus.

The structure of foot-and-mouth disease virus (FMDV) strain O1 BFS 1860 has been determined to 2.9 A resolution using the molecular-replacement method [Acharya, Fry, Stuart, Fox, Rowlands & Brown (1989). Nature (London), 337, 709-716]. Crystals of the virus with average dimensions 0.12 x 0.06 x 0.12 mm belong to space group I23, a = 345 A with 1/12 of the icosahedral particle per asymmetric unit giving fivefold noncrystallographic redundancy. Oscillation diffraction photographs were collected at the SERC Synchrotron Radiation Source at Daresbury in accordance with strict disease security regulations. The ambiguity in particle orientation was resolved using a self-rotation function and starting estimates of the phases to 8 A were derived from the known structures of two picornaviruses similarly oriented in the I23 unit cell. The phases were refined and extended using iterative averaging and solvent flattening with the implementation of a simple automatic envelope-determination procedure to increase the phasing power available.

Aphthovirus↗
PubMed1992

Crystallization and preliminary X-ray analysis of three serotypes of foot-and-mouth disease virus.

Foot-and-mouth disease viruses from serotypes O, A and C have been crystallized. The particular strains studied include O1K, A10(61), A22 Iraq 24/64, A24 Cruzeiro and C-S8c1. In addition, crystals have been grown of G67, a monoclonal antibody neutralization escape mutant derived from O1K, and of virus R100, recovered after the establishment of a persistent infection in baby hamster kidney cells with C-S8c1. Empty particles, capsids which lack the RNA genome, have also been crystallized for subtypes A22 Iraq 24/64 and A10(61). In almost all cases, crystals suitable for high resolution structure determination were obtained from (NH4)2SO4 or mixtures of polyethylene glycol and NH4Cl.

Aphthovirus↗
PubMed1992

In vitro resolution of poxvirus replicative intermediates into linear minichromosomes with hairpin termini by a virally induced Holliday junction endonuclease.

Available evidence suggests that one or more late viral gene products are involved in processing poxvirus replicative intermediates into mature progeny hairpin-terminated genomes. Cloned versions of the Shope fibroma virus (SFV) replicated telomere in the inverted repeat configuration were used as substrates to assay lysates from poxvirus-infected cells for protein fractions that participate in the resolution of the circular substrate plasmid into a linear minichromosome with viral hairpin termini. An activity in a crude protein fraction obtained from vaccinia virus-infected cells at late times during the replicative cycle was capable of accurately resolving all poxviral inverted repeat replicative intermediates tested. The resolved linear products are identical to the products of in vivo resolution and possessed symmetrical nicks which mapped at the borders of the inverted repeat sequence. Strand-specific nicks were also identified, which mapped within the telomere resolution target sequence known to be required for telomere resolution in vivo. The resolving activity that we have identified is specific to virus-infected cells at late times during replication and cleaves cloned poxviral telomeric substrates in a fashion expected of a classic Holliday junction-resolving enzyme in addition to possessing a telomere resolution target-specific nicking activity. Although a Holliday junction-resolving activity would also be expected to play a role in the recombination induced by poxvirus infection, the appearance of the activity described here only after the commencement of viral late protein synthesis suggests that it functions strictly at late times. Other non-viral Holliday junction analogs can also be cleaved by this extract, suggesting that this component of the resolution activity may also play a role in other viral processes that require cleavage of a branched DNA structure. Thus, we have identified a poxviral activity that may be a part of a protein complex which resolves concatemeric replicative intermediates of viral DNA as well as participate in general recombination late during infection.

Animals↗