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Robert D Gray

Publications and source records attributed to Robert D Gray.

9 recordsLinked to original sources
PubMed2005

Contribution of a single-turn alpha-helix to the conformational stability and activity of the alkaline proteinase inhibitor of Pseudomonas aeruginosa.

The alkaline proteinase inhibitor of Pseudomonas aeruginosa (APRin), a high-affinity inhibitor of the serralysin family of bacterial metalloproteinases, is folded into an eight-stranded beta-barrel with an N-terminal trunk linked to the barrel by a single-turn alpha-helix (helix A, residues 8-11). We show here that deletion or modification of helix A decreases the conformational stability of APRin as assessed by thermal and chemical denaturation with guanidinium chloride (GdmCl). The apparent melting temperature T(m) of the wild-type protein was 81.5 degrees C at pH 7.1 as assessed by circular dichroism and 87.5 degrees C by differential scanning calorimetry. Reduction of the single disulfide bond of APRin decreased T(m) by approximately 18 degrees C, while deletion of residues 6-10 or 1-10 lowered T(m) by approximately 8 and approximately 14 degrees C, respectively. DeltaG(u) as assessed by chemical denaturation was 7.2 kcal mol(-)(1) at 25 degrees C for wild-type APRin and was decreased by 3.4, 2.4, and 2.6 kcal mol(-)(1) by disulfide reduction, deletion of residues 6-10, and deletion of residues 1-10, respectively. In contrast, deletion of residues 1-5 had no significant effect on either T(m) or DeltaG(u). Substitution of five helix-breaking Gly or Pro residues in positions 6-10 as well as disruption of hydrogen bonds involving residues within helix A (mutants Asp10Pro and Trp15Phe) also decreased T(m) and DeltaG(u). The data suggest that a hydrogen-bonding network involving Leu11 in helix A and Trp15 located at the top of the barrel may prevent access of solvent to the interior of the barrel. Disruption of the helix could facilitate solvation of the nonpolar interior of the barrel, thereby destabilizing its folded structure. Kinetic studies with single amino acid mutants in helix A indicate that it modulates the affinity of APRin for APR primarily by influencing the dissociation rate of the inhibitor from the complex.

Amino Acid Sequence↗
PubMed2005

Decongestant effects of nasal xylometazoline and mometasone furoate in persistent allergic rhinitis.

Thirty-six persistent allergic rhinitis (PAR) sufferers were studied, to both compare and correlate 15 minute response to nasal xylometazoline (XYLO) with 28 day response to nasal mometasone furoate (MF). 0.1% XYLO (1 spray each nostril) response was measured on two occasions, then a randomised double blind cross-over comparison of MF (200 mcg daily) to placebo conducted. Outcomes were peak nasal inspiratoly flow (PNIF), nasal forced inspiratory volume in one second (nFIV1) and nasal blockage score (NBS) improvements. Thirty-one participants completed per protocol. Within subject standard deviation for percentage improvement to XYLO was 26.0 for PNIF and 25.2 for nFIV1. Median % improvement (95%CI) in PNIF for XYLO vs. MF was 20.0 (11.4 to 31.0) vs. 9.6 (3.2 to 15.8) and in nFIV1 was 17.8 (10.0 to 28.1) vs. 3.3 (-4.3 to 19.1). XYLO effects were greater than MF (p<0.05) for PNIF, nFIV1 and NBS. There was no significant correlation of MF to XYLO improvements in PNIF, nFIV1 or NBS. In conclusion, acute reversibility to XYLO showed poor repeatability and XYLO reversibility is not predictive of decongestant response to nasal corticosteroid. XYLO was a stronger decongestant than MF but rhinitis medicamentosa still precludes any preference for long term XYLO therapy at this time.

Administration, Intranasal↗
PubMed2004

Effects of butterbur treatment in intermittent allergic rhinitis: a placebo-controlled evaluation.

BACKGROUND: Butterbur (Petasites hybridus) contains the active ingredient petasin, which exhibits antileukotriene and antihistamine activity. Previous studies of intermittent allergic rhinitis (IAR) have demonstrated a comparable response to butterbur compared with a histamine H1-receptor antagonist on the 36-Item Short-Form Health Survey quality-of-life score. However, there has been no placebo-controlled study of the effects of butterbur use on objective and subjective outcomes in IAR. OBJECTIVE: To evaluate the effects of treatment with butterbur vs placebo on objective and subjective outcomes in IAR. METHODS: A double-blind, placebo-controlled, crossover study was carried out during the grass pollen season in Tayside, Scotland. Thirty-five patients (14 men and 21 women) with IAR received butterbur, 50 mg twice daily, or placebo for 2 weeks. Domiciliary measurements were taken in the morning and evening for peak nasal inspiratory flow (PNIF) (the primary outcome variable), nasal and eye symptoms, and rhinoconjunctivitis-specific quality-of-life score. RESULTS: Butterbur treatment had no significant effect on PNIF, total nasal symptom score, eye symptom score, or quality of life compared with placebo use. Mean (SEM) morning and evening PNIF values were 107 (6) and 114 (6) L/min, respectively, for butterbur vs 105 (6) and 117 (6) L/min for placebo. Mean (SEM) morning and evening total nasal symptom scores (maximum total score, 12) were 3.4 (0.4) and 3.5 (0.4), respectively, for butterbur vs 3.7 (0.3) and 3.8 (0.4) for placebo. CONCLUSIONS: There was no significant clinical efficacy of butterbur use vs placebo use on objective and subjective outcomes in IAR. Further studies are now indicated to investigate the use of butterbur in persistent allergic rhinitis.

Adult↗
PubMed2004

Single and short-term dosing effects of levocetirizine on adenosine monophosphate bronchoprovocation in atopic asthma.

AIMS: Adenosine monophosphate (AMP) acts indirectly via primed airway mast cells to induce bronchial hyper-responsiveness, which in turn correlates with eosinophilic asthmatic inflammation and atopic disease expression. We evaluated single and short-term dosing effects of a modern histamine H1-receptor antagonist, levocetirizine, given at the usual clinically recommended dose, on the primary outcome of AMP bronchoprovocation. METHODS: Fifteen atopic asthmatics were randomized in double-blind, cross-over fashion to receive for 1 week either levocetirizine 5 mg or placebo. There was a 1-week washout period prior to each randomized treatment. The provocative concentration of AMP producing a 20% fall in FEV1 (PC20) was measured after each washout at baseline and at 4-6 h following the first and last doses of each randomized treatment. RESULTS: Baseline mean +/- SEM values after washout prior to each randomized treatment comparing levocetirizine vs placebo were not significantly different for prechallenge FEV1 (% predicted) 83 +/- 4 vs 82 +/- 4, or AMP PC20 (mg ml(-1)) 45 +/- 24 vs 45 +/- 22, respectively. Airway calibre as prechallenge FEV1 for levocetirizine vs placebo was not significantly different following the first dose 86 +/- 4 vs 82 +/- 4, or the last dose 85 +/- 4 vs 83 +/- 4, respectively. There were significant improvements (P < 0.05) in AMP PC20 comparing levocetirizine vs placebo following the first dose 123 +/- 73 vs 48 +/- 24, a 1.4 doubling dilution difference (95% CI 0.8, 1.9), and the last dose 127 +/- 74 vs 53 +/- 29, a 1.2 doubling dilution difference (95% CI 0.5, 2.0). AMP PC20 was also improved (P < 0.05) by the first and last doses of levocetirizine but not placebo, vs respective baseline values, with there being no difference in the degree of protection between first and last doses. CONCLUSIONS: Single and short-term dosing with levocetirizine conferred similar improvements in bronchial hyper-responsiveness to AMP challenge, which was unrelated to prechallenge airway calibre. Further studies are indicated to evaluate the longer-term effects of levocetirizine on asthma exacerbations.

Adenosine Monophosphate↗
PubMed2003

Alkaline proteinase inhibitor of Pseudomonas aeruginosa: a mutational and molecular dynamics study of the role of N-terminal residues in the inhibition of Pseudomonas alkaline proteinase.

Alkaline proteinase inhibitor of Pseudomonas aeruginosa is a 11.5-kDa, high affinity inhibitor of the serralysin class of zinc-dependent proteinases secreted by several Gram-negative bacteria. X-ray crystallography of the proteinase-inhibitor complex reveals that five N-terminal inhibitor residues occupy the extended substrate binding site of the enzyme and that the catalytic zinc is chelated by the alpha-amino and carbonyl groups of the N-terminal residue of the inhibitor. In this study, we assessed the effect of alteration of inhibitor residues 2-5 on its affinity for Pseudomonas alkaline proteinase (APR) as derived from the ratio of the dissociation and associate rate constants for formation of the enzyme-inhibitor complex. The largest effect was observed at position Ser-2, which occupies the S1' pocket of the enzyme and donates a hydrogen bond to the carboxyl group of the catalytic Glu-177 of the proteinase. Substitution of Asp, Arg, or Trp at this position increased the dissociation constant KD by 35-, 180-, and 13-fold, respectively. Mutation at positions 3-5 of the trunk also resulted in a reduction in enzyme-inhibitor affinity, with the exception of an I4W mutant, which exhibited a 3-fold increase in affinity. Molecular dynamics simulation of the complex formation between the catalytic domain of APR and the S2D mutant showed that the carboxyl of Asp-2 interacts with the catalytic zinc, thereby partially neutralizing the negative charge that otherwise would clash with the carboxyl group of Glu-177 of APR. Simulation of the interaction between the alkaline proteinase and the I4W mutant revealed a major shift in the loop comprised of residues 189-200 of the enzyme that allowed formation of a stacking interaction between the aromatic rings of Ile-4 of the inhibitor and Tyr-158 of the proteinase. This new interaction could account for the observed increase in enzyme-inhibitor affinity.

Amino Acids↗
PubMed2003

Chronic cough.

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Adult↗