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Biomedical subjects

W R Moore

Publications and source records attributed to W R Moore.

At least 19 recordsLinked to original sources
PubMed2001

Synthetic bone graft substitutes.

Replacement of extensive local bone loss is a significant clinical challenge. There are a variety of techniques available to the surgeon to manage this problem, each with their own advantages and disadvantages. It is well known that there is morbidity associated with harvesting of autogenous bone graft and limitations in the quantity of bone available. Alternatively allografts have been reported to have a significant incidence of postoperative infection and fracture as well as the potential risk of disease transmission. During the past 30 years a variety of synthetic bone graft substitutes has been developed with the aim to minimize these complications. The benefits of synthetic grafts include availability, sterility and reduced morbidity. The present article examines the relevance of synthetic bone graft substitutes, their mechanical properties and clinical application.

Aluminum Oxide↗
PubMed2000

A novel approach to serine protease inhibition: kinetic characterization of inhibitors whose potencies and selectivities are dramatically enhanced by Zinc(II).

Serine proteases play a role in a variety of disease states and thus are attractive targets for therapeutic intervention. We report the kinetic characterization of a class of serine protease inhibitors whose potencies and selectivities are dramatically enhanced in the presence of Zn(II). The structural basis for Zn(II)-mediated inhibition of trypsin-like proteases has recently been reported [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612]. A case study of the kinetic behavior of human tryptase inhibitors is provided to illustrate the general phenomenon of Zn(II)-mediated inhibition. Tryptase, Zn(II), and the inhibitor form a ternary complex which exhibits classic tight-binding inhibition. The half-life for release of inhibitor from the tryptase-Zn(II)-inhibitor complex has been measured for a number of inhibitors. Consistent with tight-binding behavior, potent tryptase inhibitors are characterized by extremely slow rates of dissociation from the ternary complex with half-lives on the order of hours. A model of human serum, designed to reproduce physiological levels of Zn(II), has been employed to evaluate the performance of Zn(II)-potentiated tryptase inhibitors under physiological conditions. We demonstrate that Zn(II)-mediated inhibition can be achieved at physiological Zn(II) levels.

Cations, Divalent↗
PubMed1999

Neutrophil myeloperoxidase is a potent and selective inhibitor of mast cell tryptase.

Myeloperoxidase (MPO) is an important component of the neutrophil response to microbial infection. In this paper we report an additional activity of MPO, the potent and selective inhibition of human mast cell tryptase. MPO inhibits human mast cell tryptase in a time-dependent manner with an IC50 of 16 nM at 1 h. In contrast, MPO does not inhibit trypsin, thrombin, plasmin, factor Xa, elastase, or cathepsin G. It is the native protein conformation of MPO and not its enzyme activity that is responsible for tryptase inhibition. Heparin, at high concentrations, can prevent the inhibition of tryptase by MPO. We have shown by size-exclusion chromatography that MPO promotes the dissociation of active tryptase tetramer to inactive monomer. These data suggest that MPO inhibits tryptase by interfering with the heparin stabilization of tryptase tetramer. We have previously shown that lactoferrin (another neutrophil-associated protein) also inhibits tryptase activity by a similar mechanism. The finding that MPO is a potent inhibitor of tryptase lends further support to the hypothesis that neutrophil proteins, such as MPO and lactoferrin, may play a regulatory role as endogenous suppressers of tryptase enzyme activity.

Chymases↗
PubMed1998

Design of potent selective zinc-mediated serine protease inhibitors.

Many serine proteases are targets for therapeutic intervention because they often play key roles in disease. Small molecule inhibitors of serine proteases with high affinity are especially interesting as they could be used as scaffolds from which to develop drugs selective for protease targets. One such inhibitor is bis(5-amidino-2-benzimidazolyl)methane (BABIM), standing out as the best inhibitor of trypsin (by a factor of over 100) in a series of over 60 relatively closely related analogues. By probing the structural basis of inhibition, we discovered, using crystallographic methods, a new mode of high-affinity binding in which a Zn2+ ion is tetrahedrally coordinated between two chelating nitrogens of BABIM and two active site residues, His57 and Ser 195. Zn2+, at subphysiological levels, enhances inhibition by over 10(3)-fold. The distinct Zn2+ coordination geometry implies a strong dependence of affinity on substituents. This unique structural paradigm has enabled development of potent, highly selective, Zn2+-dependent inhibitors of several therapeutically important serine proteases, using a physiologically ubiquitous metal ion.

Animals↗
PubMed1998

Inhibitors of tryptase for the treatment of mast cell-mediated diseases.

Human tryptase is a structurally unique and mast cell specific trypsin-like serine protease. Recent biological and immunological investigations have implicated tryptase as a mediator in the pathology of numerous allergic and inflammatory conditions including rhinitis, conjunctivitis, and most notably asthma. A growing body of data further implicates tryptase in certain gastrointestinal, dermatological, and cardiovascular disorders as well. The recent availability of potent, and selective tryptase inhibitors, though, has facilitated the validation of this protease as an important therapeutic target as well. Herein, we describe the design and potency of four classes of selective tryptase inhibitors, of which the first three types are synthetic and the fourth is natural in origin: 1) peptidic inhibitors (e.g., APC-366), 2) dibasic inhibitors (i.e., pentamidine-like), 3) Zn(2+)-mediated inhibitors (i.e., BABIM-like), and 4) heparin antagonists (e.g., lactoferrin). These inhibitors have been tested in the airways and skin of allergic sheep. Aerosol administration of tryptase inhibitors from each structural class 30 minutes before, and 4 hours and 24 hours after allergen challenge, abolishes late phase bronchoconstriction and airway hyperresponsiveness in a dose-dependent manner. Moreover, intradermal injection of APC-366 blocks the cutaneous response to antigen. These studies provide the essential proof-of-concept for the further pursuit of tryptase inhibitors for the treatment of asthma, and perhaps other allergic diseases. Results from clinical studies with the first generation tryptase inhibitor APC-366, currently in phase II trials for the treatment of asthma, provide additional support for a pathological role for tryptase in this disease. Notable advances in the area of tryptase inhibitor design at Axys Pharmaceuticals, Inc. include a novel, zinc-mediated, serine protease inhibitor technology (described herein), and the discovery of a unique class of extremely potent and selective dibasic tryptase inhibitors. Independently, an X-ray crystal structure of active tryptase tetramer complexed with 4-amidinophenyl pyruvic acid has been reported. It is anticipated that these discoveries will further accelerate the design of structurally novel tryptase inhibitors as well as the development of new drugs for the treatment of mast cell tryptase-mediated disorders.

Animals↗
PubMed1997

Lactoferrin, a potent tryptase inhibitor, abolishes late-phase airway responses in allergic sheep.

Tryptase, a serine protease released exclusively from activated mast cells, has been implicated as a potential causative agent in asthma. Enzymatically active tryptase is comprised of four subunits, and heparin stabilizes the associated tetramer. Lactoferrin, a cationic protein released from activated neutrophils, binds tightly to heparin, therefore we investigated lactoferrin as an inhibitor of tryptase and found that it is both a potent (Ki' is 24 nM) and selective inhibitor. Size exclusion chromatography studies revealed that lactoferrin disrupted the quaternary structure of active tryptase. Lactoferrin was tested in an allergic sheep model of asthma; aerosolized lactoferrin (10 mg in 3 ml phosphate-buffered saline, 0.5 h before as well as 4 and 24 h after inhalation challenge by Ascaris suum) abolished both late-phase bronchoconstriction (no significant increase in specific lung resistance 4 to 8 h following provocation, p < 0.05 versus vehicle treatment) and airway hyperresponsiveness (no detectable increase in airway sensitivity to carbachol challenge 24 h after antigen challenge, p < 0.05 versus vehicle). These data suggest tryptase involvement in both late-phase bronchoconstriction and airway hyperreactivity and furthermore suggest that a physiological function of neutrophil lactoferrin is the inhibition of tryptase released from mast cells.

Airway Resistance↗
PubMed1996

(-) mating type-specific mutants of Phycomyces defective in sex pheromone biosynthesis.

We have isolated the first mating type-specific mutants in mucoraceous fungi. Both mutants in Phycomyces blakesleeanus appear to be defective in the same gene. The gene, present in both mating types, is necessary only in cultures of the (-) mating type. The gene codes for an enzyme in sex pheromone biosynthesis. The pheromone precursor made by the mutants is detectable only in cross-feeding experiments. The biological and solubility properties of the precursor suggest the precursor is 4-dihydrotrisporin, a metabolite of beta-carotene. Separate studies with beta-carotene-deficient mutants and Compound-P, a new chemically synthesized precursor of the pheromones, imply the constitutive level of enzymes for pheromone biosynthesis in Phycomyces is extremely low. In comparison, the level of enzymes for pheromone conversion to trisporic acid is higher. The mating type-specific mutants also catalyze the conversion of (+) pheromone to trisporic acid. This finding was unexpected because literature models predicted this reaction was catalyzed by the same enzyme which catalyzed the conversion of 4-dihydrotrisporin to (-) pheromone-a reaction missing in the (-) mating type-specific mutants. Thus, we propose a revised model for trisporic acid biosynthesis.

Crosses, Genetic↗
PubMed1996

Inhaled tryptase causes bronchoconstriction in sheep via histamine release.

Allergen-induced bronchoconstriction involves mast cell activation. Tryptase is a mast cell serine protease that is released during this process, but little is known about the action of tryptase in the airway. The purpose of this study was to determine: (1) if aerosolized tryptase causes bronchoconstriction, and (2) the mechanism by which this occurs. We measured mean pulmonary flow resistance (RL) in five allergic sheep before and after consecutive inhalations of 100 and 500 ng tryptase (in 2 ml total volume). Inhaled tryptase at 100 and 500 ng increased RL (mean +/- SE) by 33 +/- 12 and 122 +/- 8% (p < 0.05) over baseline. The response was reproducible upon repeat challenges. These studies were repeated in the same animals after pretreatment with aerosolized APC 366 (9 mg/3 ml), a specific tryptase inhibitor. In APC-366-treated sheep, tryptase increased RL by 10 +/- 3 and 6 +/- 2% (p < 0.05 versus control values) at 100 and 500 ng, respectively. The response to tryptase was also blocked by pretreating the sheep intravenously with the histamine H1-antagonist chlorpheniramine (2 mg/kg), in which RL increased only 5 +/- 4 and 7 +/- 6% after 100 and 500 ng tryptase. APC 366, however, did not block histamine-induced bronchoconstriction. Consistent with these findings was the observation that segmental bronchial challenge with tryptase (1 microgram) resulted in a significant increase in histamine levels in bronchoalveolar lavage. Inhaled tryptase (500 ng) also caused airway hyperresponsiveness to aerosolized carbachol 2 h after tryptase challenge. This tryptase-induced airway hyperresponsiveness could be blocked either by pretreating the sheep with APC 366 (30 min before challenge) or by treating the sheep 30 min after challenge. These results indicate that inhaled tryptase causes bronchoconstriction and airway hyperresponsiveness in allergic sheep by an event that may involve mast cell activation.

Administration, Inhalation↗
PubMed1995

Role of tryptase in immediate cutaneous responses in allergic sheep.

In this study, we used a specific tryptase inhibitor, APC-366 [N-(1-hydroxy-2-napthoyl)-L-arginyl-L- prolinamide hydrochloride] to investigate the effect of intradermally administered tryptase and tryptase released by antigen challenge on the immediate cutaneous reaction (ICR) in allergic sheep. The surface areas of cutaneous wheals produced by intradermal injections (0.05 ml) of 1 and 10 ng tryptase alone, tryptase combined with 3 U heparin (tryptase-heparin), or Ascaris suum antigen (10(-5) dilution) with or without pretreatment with APC-366 (1 mg/ml) were measured at 20 and 60 min after challenge. Intradermal injections of 1 and 10 ng tryptase alone (n = 7) produced an ICR of < or = 20% of that obtained after injection of histamine (5% wt/vol). Intradermal injection of tryptase-heparin (n = 7), however, resulted in 50 (1 ng) and 82% (10 ng) of the ICR to histamine (both, P < 0.05 vs. tryptase alone). APC-366 inhibited (P < 0.05) the ICR to 1 and 10 ng tryptase-heparin by > or = 70% at all times (n = 8) but had no effect on the histamine-induced ICR (n = 3). A combination of the histamine H1 antagonist chlorpheniramine (2 mg/kg iv) and the H2 antagonist metiamide (3 mg/kg iv) given 40 min before challenge (n = 8) inhibited the response to 1 and 10 ng tryptase-heparin by 42 and 62% at 20 min and by 96 and 86% at 60 min, respectively (all, P < 0.05). APC-366 also blocked the ICR to A. suum antigen by 68% (P < 0.05) in nine sheep. These results indicate that intradermal injection of tryptase-heparin can induce an ICR. This ICR can be inhibited by APC-366 or a combination of the histamine H1 and H2 antagonists, suggesting that the tryptase response is mediated by histamine. APC-366 also blocks the mast cell-mediated ICR to intradermally injected A. suum antigen. Collectively, these results suggest that tryptase may modulate mast cell histamine release.

Animals↗
PubMed1995

Tryptase inhibitors block allergen-induced airway and inflammatory responses in allergic sheep.

Tryptase, a mast cell serine protease, has been implicated in the pathophysiology of allergic asthma, but formal evidence to support this hypothesis has been limited by the lack of specific inhibitors for use in vivo. Therefore, in this study we examined the effects of two inhibitors of tryptase, APC 366 [N-(1-hydroxy-2-naphthoyl)-L-arginyl-L-prolinamide hydrochloride] and BABIM [bis(5-amidino-2-benzimidazolyl)methane] on antigen-induced early and late responses, airway responsiveness as measured by carbachol provocation, microvascular permeability as measured by bronchoalveolar lavage (BAL) albumin concentrations, and tissue eosinophilia from biopsies in allergic sheep. APC 366 and BABIM were administered by aerosol in all experiments. In vehicle control trials, antigen challenge resulted in peak early and late increases in specific lung resistance (SRL) of (mean +/- SE, n = 6) 259 +/- 30% and 183 +/- 27% over baseline, respectively. Treatment with APC 366 (9 mg/3 ml H2O given 0.5 h before, 4 h after, and 24 h after antigen challenge) slightly reduced the peak early response (194 +/- 41%), but significantly inhibited the late response (38 +/- 6%, p < 0.05 versus control trials). Twenty-four hours after challenge, APC 366 also completely blocked the antigen-induced airway hyperresponsiveness to inhaled carbachol observed in the control trial.(ABSTRACT TRUNCATED AT 250 WORDS)

Airway Resistance↗
PubMed1992

Purification of 2,3-oxidosqualene cyclase from rat liver.

A rapid and simple purification of milligram amounts of 2,3-oxidosqualene cyclase, an integral membrane enzyme that catalyzes the cyclization of squalene epoxide to lanosterol, is reported. Several nonionic detergents (Triton X-100, Tween 80, Emulphogene, and lauryl maltoside) were evaluated for solubilization of oxidosqualene cyclase from rat liver microsomes. At a detergent concentration of 5 mg/ml, lauryl maltoside was approximately 10 times more effective than Emulphogene in the solubilization of oxidosqualene cyclase; Triton X-100 and Tween 80 were less effective than Emulphogene as judged by the relative specific activities of the solubilized enzyme. Treatment of microsomes with lauryl maltoside resulted in a selective solubilization of the cyclase with concomitant activation of the enzyme. The solubilized enzyme was purified to homogeneity by fast protein liquid chromatography. The purified enzyme consists of a single subunit that has an apparent molecular weight of 65,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme obeys saturation kinetics and the apparent Km of (2,3)-oxidosqualene is 15 microM; the apparent kcat/Km is 200 M-1.min-1. An improved assay of the enzyme that utilizes high performance liquid chromatography methods is also described.

Animals↗
PubMed1992

The purification and properties of cancer procoagulant from murine tumors.

The protease, cancer procoagulant, was isolated from three murine metastatic tumors and was purified to apparent homogeneity (SDS-PAGE) from Lewis lung cells by the sequence of (NH4)2SO4 precipitation, DE-53 anion-exchange chromatography, and Sephacryl 200 chromatography. The murine tumor enzyme has a molecular weight of 68,000 and Ca2+ is required for procoagulant and proteolytic activity; thus, the murine enzyme is very similar to that isolated from rabbit tumors. Two peptidyl chromogenic substrates of cancer procoagulant were discovered, facilitating kinetic and inhibition studies with the enzyme. The peptide substrate structures and the results of inhibition studies suggest that cancer procoagulant is thrombin-like in specificity but is a thiol protease.

Animals↗
PubMed1991

Contrast cholangiography versus ultrasonographic measurement of the "extrahepatic" bile duct: a two-fold discrepancy revisited.

The upper limit of the normal extrahepatic duct diameter when measured by sonography in our institution is less than half that when measured by endoscopic retrograde cholangiopancreatography (ERCP). The objective of this study was to locate possible sources of this discrepancy by comparing measurements obtained on the same patient by sonography, before and after ERCP. Thirty consecutive patients referred for ERCP were entered into a prospective trial; bile duct measurements were obtained independently by both techniques in 19 patients. Correcting for radiographic magnification, the ERCP measurement was more than twice that obtained by ultrasonography. Among the possible reasons for diverse results that we considered, radiographic magnification, ultrasonic underestimation, and distending effects of retrograde cholangiography (ERCP) were not found to be prominent causes for the marked discrepancy that we observed. The most likely explanation is that the duct in individual patients was being measured at a different level by the two techniques. A retrospective comparison of the studies obtained by each method suggests that the sonographic measurement is most often of the right hepatic duct. If this is the case, the two measurements show no statistically significant difference (P greater than 0.05). A prospective trial is needed to test this hypothesis.

Bile Ducts↗
PubMed1989

Increased capacity for glutathione synthesis enhances resistance to radiation in Escherichia coli: a possible model for mammalian cell protection.

A strain of Escherichia coli, enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase activities by recombinant DNA techniques, is more resistant to the lethal effects of gamma-irradiation than is the corresponding wild strain. Although the gene-enriched strain has higher glutathione levels than the wild strain, the observed radioresistance appears to be associated with the increased capacity of the gene-enriched strain to synthesize glutathione when irradiated rather than to the cellular levels of glutathione per se. Thus, resistance was abolished in the presence of buthionine sulfoximine, a selective inactivator of gamma-glutamylcysteine synthetase that decreases glutathione synthesis but that does not act directly to lower cellular glutathione levels. Conclusions drawn from studies on this E. coli model system may have relevance to protection of mammalian cells by glutathione.

Antimetabolites↗
PubMed1988

On the active site thiol of gamma-glutamylcysteine synthetase: relationships to catalysis, inhibition, and regulation.

gamma-Glutamylcysteine synthetase (glutamate-cysteine ligase; EC 6.3.2.2) was isolated from an Escherichia coli strain enriched in the gene for this enzyme by recombinant DNA techniques. The purified enzyme has a specific activity of 1860 units/mg and a molecular weight of 56,000. Comparison of the E. coli enzyme with the well-characterized rat kidney enzyme showed that these enzymes have similar catalytic properties (apparent Km values, substrate specificities, turnover numbers). Both enzymes are feedback-inhibited by glutathione but not by gamma-glutamyl-alpha-aminobutyrylglycine; the data indicate that glutathione binds not only at the glutamate binding site but also at a second site on the enzyme that interacts with the thiol moiety of glutathione but not with a methyl group. Both enzymes are inactivated by buthionine sulfoximine in the presence of ATP, suggesting a common gamma-glutamyl phosphate intermediate. However, unlike the rat kidney enzyme that has an active center thiol, the bacterial enzyme is insensitive to cystamine, gamma-methylene glutamate, and S-sulfo amino acids, indicating that it does not have an active site thiol. Thus, the rat kidney and E. coli enzymes share several catalytic features but differ in active site structure. If the active site thiol of the rat kidney enzyme is involved in catalysis, which seems likely, there would appear to be differences in the mechanisms of action of the two gamma-glutamylcysteine synthetases.

Animals↗
PubMed1987

Enzymatic synthesis of novel glutathione analogs.

A strain of Escherichia coli enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase by recombinant DNA techniques has been immobilized in a carrageenan matrix and used for the synthesis of various types of isotopically labeled glutathione (L-gamma-glutamyl-L-cysteinyl-glycine) (K. Murata, W. A. Abbott, R. J. Bridges, and A. Meister (1985) Anal. Biochem. 150, 235-237). In the present work, this E. coli matrix was used as the basis of a method for the synthesis of glutathione analogs. Thus, amino acid analogs were used in place of the corresponding amino acid constituents of glutathione (e.g., 4-fluoroglutamate was substituted for glutamate) in the reaction mixtures. Using this method we have synthesized several analogs of glutathione including L-gamma-glutamyl-(beta-chloro)-L-alanyl-glycine, (R,S)-4-fluoro-DL-gamma-glutamyl-L-cysteinyl-glycine, D-gamma-glutamyl-L-cysteinyl-glycine, and L-gamma-glutamyl-L-homocysteinyl-glycine. This method may also be used for the synthesis of a number of L- and D-gamma-glutamyl amino acids. The analogs are purified by gel-filtration and ion-exchange chromatography. The analogs are used to examine the substrate specificity and mechanisms of action of glutathione-utilizing enzymes and for studies on glutathione metabolism and function. Fluorine-containing analogs may be used for NMR studies. The enzymatically prepared compounds may also be used as intermediates in the chemical synthesis of other analogs of glutathione and glutathione disulfide.

Amino Acids↗
PubMed1986

Caustic ingestions. Pathophysiology, diagnosis, and treatment.

More than 5,000 caustic ingestions are estimated to occur each year. Liquid lye is associated with the greatest morbidity. Endoscopy is the only method for evaluating the extent of the injury; clinical signs and symptoms (e.g., vomiting, dysphagia, abdominal pain, oral burns, leukocytosis) are not reliable. In less severe burns (involving only mucosal irritation or ulceration), current therapy with antibiotics and steroids results in a very favorable prognosis. Circumferential or very deep burns have a much poorer outcome, with a greatly increased risk of stricture formation and/or perforation. The risk of developing esophageal carcinoma may be 1,000 times greater for individuals who have had a lye burn than in the general population. It cannot be overemphasized that prevention is still the key to decreasing this morbidity.

Ampicillin↗