Search PubMed⌕ Search

Biomedical subjects

J D Stewart

Publications and source records attributed to J D Stewart.

At least 37 records · Page 2Linked to original sources
PubMed1999

Tryptophan H33 plays an important role in pyrimidine (6-4) pyrimidone photoproduct binding by a high-affinity antibody.

The importance of Trp H33 in antibody recognition of DNA containing a central pyrimidine (6-4) pyrimidone photoproduct was investigated. This residue was replaced by Tyr, Phe and Ala and the binding abilities of these mutants were determined by surface plasmon resonance and fluorescence spectroscopy. Conservative substitution of Trp H33 by Tyr or Phe resulted in moderate losses of binding affinity; however, replacement by Ala had a significantly larger impact. The fluorescence properties of DNA containing a (6-4) photoproduct were strongly affected by the identity of the H33 residue. DNA binding by both the wild-type and the W-H33-Y mutant was accompanied by a small degree of fluorescence quenching; by contrast, binding by the W-H33-F and W-H33-A mutants produced large fluorescence increases. Taken together, these variations in binding and fluorescence properties with the identity of the H33 residue are consistent with a role in photoproduct recognition by Trp H33 in the high-affinity antibody 64M5.

Alanine↗
PubMed1999

Effects of a high-affinity antibody fragment on DNA polymerase reactions near a (6-4) photoproduct site.

Pyrimidine (6-4) pyrimidone photodimers are major photoproducts that have mutagenic and carcinogenic consequences. One major reason for these biological effects of (6-4) photoproducts may be base mispairing/DNA replication errors due to hydrogen bonding to bases opposite these damaged sites. We synthesized a modified 41-mer DNA containing a (6-4) photoproduct using a preformed building block, then employed it as a template for primer extension reactions catalyzed by Klenow fragment and DNA polymerases alpha, beta and delta (pol alpha, pol beta and pol delta). None of these DNA polymerases were able to bypass the (6-4) photoproduct and elongation terminated at or near the 3'-pyrimidone of the photoproduct, depending on the dNTP concentration. When a single-chain Fv (scFv) with high affinity for the (6-4) photoproduct was included in the polymerization reaction, DNA synthesis was inhibited at base positions four, six, eight or eight nucleotides prior to the 3'-pyrimidone by Klenow fragment, pol alpha, pol beta or pol delta, respectively. These results suggest that the scFv can bind to the template DNA containing a (6-4) photoproduct and inhibit extension reactions by polymerases.

Animals↗
PubMed1998

Kinetic mechanism of Escherichia coli asparagine synthetase B.

Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartate, glutamine, and ATP. A combination of kinetic, isotopic-labeling, and stoichiometry studies have been performed to define the nature of nitrogen transfer mediated by AS-B. The results of initial rate studies were consistent with initial binding and hydrolysis of glutamine to glutamate plus enzyme-bound ammonia. The initial velocity results were equally consistent with initial binding of ATP and aspartate prior to glutamine binding. However, product inhibition studies were only consistent with the latter pathway. Moreover, isotope-trapping studies confirmed that the enzyme-ATP-aspartate complex was kinetically competent. Studies using 18O-labeled aspartate were consistent with formation of a beta-aspartyl-AMP intermediate, and stoichiometry studies revealed that 1 equiv of this intermediate formed on the enzyme in the absence of a nitrogen source. Taken together, our results are most consistent with initial formation of beta -aspartyl-AMP intermediate prior to glutamine binding. This sequence leaves open many possibilities for the chemical mechanism of nitrogen transfer.

Adenosine Monophosphate↗
PubMed1998

Antibodies specific for (6-4) DNA photoproducts: cloning, antibody modeling and construction of a single-chain Fv derivative.

We have investigated a series of four monoclonal antibodies that specifically recognize pyrimidine (6-4) pyrimidone photoproducts. One of these antibodies (64M4), bound all four possible pyrimidine-pyrimidone photoadducts with equal affinities whereas the others (64M2, 64M3 and 64M5) were selective for TC and TT sequences. In addition, 64M5 had the highest binding affinity for photodamaged DNA of the four [T. Mori et al., Photochem. Photobiol. 54 (1991) 225-232]. To help understand the differences between these antibodies, we have cloned and sequenced the variable region genes from all four. Comparing these sequences revealed that all four were highly similar to one another, although there were some differences in potential antigen-contact regions. To assess the influences of these sequence differences at the structural level, computer models were constructed for all four antibodies. Most of the sequence differences occurred in potential antigen contact regions, suggesting specific positions that might account for the observed differences in binding affinities and selectivities. A single-chain Fv derivative of 64M5 was therefore constructed and characterized to provide an experimental system in which structure-function relationships can be tested. This derivative could be isolated from Escherichia coli using two chromatographic steps and possessed the same binding specificity as the parent monoclonal antibody.

Amino Acid Sequence↗
PubMed1998

Normal distributions of thermal and vibration sensory thresholds.

The distributions of sensory thresholds were estimated in a healthy population while controlling for potential covariates. Using the method of levels and the two-alternative forced choice, thermal and vibration thresholds respectively were measured in the hand and foot of 148 subjects. Age was uniformly distributed between 20 and 86 years. Independent effects of age, gender, height, and skin temperature were estimated using multiple linear regression. Parametric and nonparametric methods were used to estimate the distributions of interest. Significant age-related increases were observed for all vibration thresholds (P < 0.0001), and for thermal thresholds in the foot (P < 0.0002). Percentiles were estimated for thermal thresholds in the hand and age-adjusted continuous distributions were calculated for all other thresholds. Height was positively associated with vibration thresholds in the foot (P < 0.003), and appropriate corrections were made. Our results provide reference values for thermal and vibration sensory thresholds in a healthy population, allowing for the accurate diagnosis of disordered sensory function.

Adult↗
PubMed1998

Effectiveness of prostaglandin E2 intracervical gel (Prepidil), with immediate oxytocin, versus vaginal insert (Cervidil) for induction of labor.

OBJECTIVE: Our purpose was to compare the effectiveness of labor induction with use of prostaglandin E2 either as an intracervical gel (Prepidil), with immediate oxytocin, or as a sustained-release vaginal insert (Cervidil) with subsequent oxytocin as needed. STUDY DESIGN: Hospitalized patients at >/=37 weeks' gestation requiring labor induction and having an unfavorable cervix (Bishop score </=6) were randomly assigned to receive either Prepidil or Cervidil. Oxytocin was begun immediately after Prepidil placement or 30 minutes after removal of the Cervidil insert if needed. RESULTS: Of the 150 patients, there were no differences in demographics and eventual pregnancy outcomes between the Prepidil group (n = 77) and the Cervidil group (n = 73). Those pregnancies receiving the Prepidil-immediate oxytocin regimen were delivered sooner than those receiving the Cervidil among nulliparous (11.3 +/- 7.3 hours vs 25.2 +/- 12.5 hours, P <.001) and multiparous (8.4 +/- 7.8 hours vs 18.4 +/- 7.2 hours, P <.001) women. The mean cost savings, which favored the Prepidil-immediate oxytocin regimen, was $458 (range $204 to $630) per patient. CONCLUSION: Compared with Cervidil, the Prepidil-immediate oxytocin regimen resulted in a shorter induction-to-vaginal delivery interval and in more hospital cost savings without increasing adverse outcomes.

Administration, Intravaginal↗
PubMed1998

Pre-eclampsia and induction of labor: a randomized comparison of prostaglandin E2 as an intracervical gel, with oxytocin immediately, or as a sustained-release vaginal insert.

OBJECTIVE: Our purpose was to compare the efficacy of commercial prostaglandin E2 products, in combination with oxytocin, for the initiation of labor among pregnancies with pre-eclampsia. STUDY DESIGN: Patients with pregnancy-induced hypertension and with either proteinuria or other end-organ damage were enrolled if they had an unfavorable Bishop score (</=4) and were eligible to undergo labor. Each was randomly assigned to receive prostaglandin E2 either as a 0. 5-mg intracervical gel (Prepidil) or as a 10-mg controlled-release vaginal insert (Cervidil). Oxytocin was begun either immediately after instillation of the gel or was delayed until after removal of the insert. RESULTS: Of the 70 patients, there were no differences between the Prepidil (n = 34) and the Cervidil (n = 36) groups in maternal demographics, gestational age, parity, and predose Bishop score. There was a mean 14.3-hour difference in the duration from beginning therapy until vaginal delivery in the Prepidil group than in the Cervidil group (11.5 +/- 2.3 hours vs 25.8 +/- 6.9 hours, P <. 001). This time difference, which favored use of Prepidil-immediate oxytocin, remained significant after parity (nulliparous: 20 hours, P <.005; multiparous: 12 hours, P <.01) and gestational age were controlled (preterm: 15.5 hours, P <.01; term: 13.3 hours, P <.01). CONCLUSION: Use of combined intracervical prostaglandin E2 gel-immediate oxytocin therapy was more effective in shortening the induction-to-vaginal delivery time than use of a controlled-release prostaglandin E2 vaginal insert.

Administration, Intravaginal↗
PubMed1998

Placebo-controlled comparison between a single dose and a multidose of betamethasone in accelerating lung maturation of mice offspring.

OBJECTIVE: Our purpose was to determine, in a placebo-controlled manner with a mouse model, whether a multidose of betamethasone is more beneficial than a single dose in accelerating fetal lung maturation. STUDY DESIGN: Ninety gravid CD-1 mice were randomly assigned to 1 of 3 groups (n = 30) to receive either a placebo (0.25 mL subcutaneously) or betamethasone (0.1 mg subcutaneously) as a single dose on gestational day 14 or as a multidose twice daily on gestational day 14 and 15. Ten pregnancies in each group were terminated at gestational day 16.5 to observe the neonatal breathing pattern (scale 0 to 5; 5 is unlabored breathing) and the lung histologic findings (scale 0 to 5; 5 is alveolar budding). The lungs of the offspring belonging to the remaining 20 pregnancies in each group were removed and weighed at postnatal day 1, 3, 5, or 120. RESULTS: Fetuses exposed to a multidose of betamethasone displayed a higher breathing score at gestational day 16.5 than either to a single dose or to the placebo (mean score 4.6 vs 3.8 or 1.3; P <. 001). Alveolar development was greater after exposure to a multidose of betamethasone than after a single dose or after a placebo (mean score 4.4 vs 3.5 or 1.6; P <.001). The lung weights at gestational day 16.5 were less after a multidose of betamethasone than after a single dose of either betamethasone or a placebo (18.3 +/- 1.0 g vs 21.4 +/- 1.3 g or 23.3 +/- 1.3 g; P <.02). The lung/body weight ratio was similarly affected. This reduced weight of the lungs persisted postnatally into adulthood. CONCLUSIONS: With a CD-1 mouse model, a multidose of antenatal betamethasone accelerated fetal lung maturation more than after a single dose but was accompanied with a decrease in lung weight that persisted into adulthood.

Animals↗
PubMed1998

The role of surface lysines in pyrimidine (6-4) pyrimidone photoproduct binding by a high-affinity antibody.

The roles of four VH surface lysine side-chains in antibody recognition of DNA containing a central pyrimidine (6-4) pyrimidone photoproduct was investigated. Three of the four lysines (at positions H62, H64 and H66) are strongly conserved; however, the presence of lysine at position H68 was highly unusual. Each lysine was replaced individually by alanine; in addition, a quadruple mutant was also created. Studies of the binding kinetics revealed that these lysine residues mainly influenced the association phase. Our results suggest that these side chains help to guide the DNA polyanion to the antigen binding pocket by electrostatic effects.

Antibody Affinity↗
PubMed1998

Detection and typing of the virulence determinants cagA and vacA of Helicobacter pylori directly from biopsy DNA: are in vitro strains representative of in vivo strains?

BACKGROUND: The relationship of Helicobacter pylori genotypes to gastrointestinal disease has relied on cultured isolates. This assumes that cultured strains are representative of in vivo strains. OBJECTIVE: To detect and type the cagA status and the vacA genotypes directly from biopsy DNA without the need for culture, and to further define the relationship between H. pylori genotypes and gastroduodenal pathology. METHODS: Fifty-two Caucasian patients undergoing routine endoscopy for dyspepsia had additional biopsies taken from four gastric sites and one duodenal site for biopsy DNA preparation. An antral sample was taken for biopsy culture. All recruited patients were H. pylori-positive on rapid urease test for Campylobacter-like organisms (CLO test) and/or histology. By polymerase chain reaction (PCR), the cagA status and the vacA s and m types were detected directly from biopsy DNA. RESULTS: H. pylori isolates were cultured from 28/52 patients in whom infection was detected by PCR. Two isolate types differed from biopsy types. Fifty of the 52 patients, strains were typable from all four gastric sites and in 51/52 the same strain predominated throughout. The cancer strains were all cagA-positive/vacA s1 type. There was a correlation between cagA positivity and vacA s1 (41/43). There was no difference between the cagA-positive/vacA s1 strains and the presence or absence of ulcers. There were only 5/52 vacA s2 m2 and four were in the non-ulcer dyspeptic group. CONCLUSION: cagA status and the vacA genotyping was successful with tissue samples taken directly from gastric and duodenal biopsies. Discrepancies between isolate and biopsy strain types stress the need for caution when interpreting in vitro strain types and suggest that direct PCR of biopsies is the preferred typing technique. The cagA status and the s1 vacA allele are unreliable as single markers in determining the risk of developing peptic ulcer disease.

Antigens, Bacterial↗
PubMed1997

Impact of multiple antenatal doses of betamethasone on growth and development of mice offspring.

OBJECTIVE: Our purpose was to determine in a randomized, placebo-controlled manner whether multiple antenatal doses of betamethasone affect long-term growth and development of exposed mouse offspring. STUDY DESIGN: Sixty pregnant CD-1 mice received either two, four, or eight antepartum doses of 0.1 mg betamethasone or placebo. Perinatal outcomes, growth, and development of the offspring were compared in a blinded manner. Variables were compared by analysis of variance or chi 2 testing. RESULTS: Betamethasone-exposed subjects gained less weight during pregnancy and were delivered of fewer live pups, with fewer male survivors and lower birth weights. These trends were dose related. Growth measurements were similar after the neonatal period. No differences in functional development and physical maturation in the offspring were noted. The reproductive capability, perinatal outcomes, and growth and development of the second-generation offspring were unaffected by betamethasone exposure. CONCLUSION: Multiple antenatal dosings of betamethasone, reaching toxic levels, did not have an impact on the long-term growth and development of the surviving mouse offspring.

Animals↗
PubMed1996

Chronic inflammatory demyelinating polyneuropathy (CIDP) in diabetics.

Seven diabetic patients developed a progressive, moderately severe, motor rather than sensory neuropathy predominantly affecting the legs. This met clinical and electrophysiological criteria for chronic inflammatory demyelinating polyneuropathy (CIDP). Nerve biopsies showed a variety of abnormalities, none of which clearly distinguished between diabetic polyneuropathy and CIDP. The patients were treated with combinations of corticosteroids, azathioprine, plasmapheresis and intravenous immune globulin; all improved substantially. We believe that CIDP may masquerade as unusually severe and progressive diabetic distal symmetric polyneuropathy. It is important to recognize CIDP in diabetics because, unlike diabetic polyneuropathy, CIDP is treatable.

Adult↗
PubMed1995

Undergraduate training in laboratory animal science: a challenge for change.

LABORATORY animals are considered to be an 'obscure' specialist area by many veterinary surgeons, despite the fact that the knowledge they apply in their daily work and all of the medicines they use depend on research or testing using laboratory animals. The employment of veterinary surgeons in research is a well known aspect of the profession. Less well known is the contribution made by veterinarians as toxicological pathologists, assessing the effects of new chemicals and pharmaceuticals on laboratory animals used in safety testing. But the most important contribution of the profession is as advisers on laboratory animal welfare and health. The British Laboratory Animals Veterinary Association (BLAVA) is a specialist division of the BVA dedicated to representing and educating the profession in this area. Since 1986 every research institute has had to employ a named veterinary surgeon to advise on the health and welfare of these animals and BLAVA has therefore grown to over 200 members. In this paper BLAVA sets out its vision of how undergraduate education could meet this challenge.

Animals↗
PubMed1995

A chemiluminescent immunoassay for the identification of functional single-chain antibodies.

Random mutagenesis of a template single-chain Fv (scFv) antibody generates a mutant library with members of varying ligand affinities; however, only a small population of these mutants exhibit strong (Kd approximately 1 nM) or improved affinities. We have therefore designed a chemiluminescent sandwich assay which can identify strong ligand-binding mutants expressed by Escherichia coli. A model system involving scFv 43C9 and selected mutants with varying hapten affinities was used to demonstrate the application of this methodology. Both the plaque and colony lifts of these scFvs were screened for hapten-binding activities with the chemiluminescent assay, and only strong binders were detected.

Antibodies, Bacterial↗
PubMed1995

Transition-state stabilization as a measure of the efficiency of antibody catalysis.

There are now about 60 examples of reactions that have been successfully catalysed by monoclonal antibodies. Not surprisingly, many of the early examples involved reactions that were already favoured kinetically (such as carbonate and ester hydrolysis). But it has since been shown that antibodies can also accelerate reaction pathways that are normally disfavoured kinetically (by at least a few kcal mol-1). Here we use transition-state theory to provide a quantitative analysis of the scope and limitations of antibody catalysis. We show that the observed rate accelerations can be predicted from the ratio of equilibrium binding constants of the reaction substrate and the transition-state analogue used to raise the antibody. This scheme allows us to rationalize the product selectivity displayed in antibody catalysis of disfavoured reactions, to predict the degree of rate acceleration that catalytic antibodies may ultimately afford, and to highlight some differences between the way that they and enzymes catalyse reactions.

Antibodies, Catalytic↗