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W L Smith

Publications and source records attributed to W L Smith.

At least 145 records · Page 8Linked to original sources
PubMed1994

Interactions of PGH synthase isozymes-1 and -2 with NSAIDs.

There are two isozymes of prostaglandin endoperoxide (PGH) synthase (cyclooxygenase) called PGH synthase-1 and -2 or COX I and II. Both isozymes catalyze the same two reactions: oxygenation of arachidonate to yield PGG2 and reduction of PGG2 to PGH2. PGH synthase-1 is expressed constitutively and is found in most tissues. PGH synthase-2 is undetectable in most cells but can be induced in fibroblasts, endothelial cells, ovarian follicles, and macrophages by various mitogens, cytokines, and tumor promoters. PGH synthase-1 (PGHS-1) has been presumed to be the site of action of nonsteroidal antiinflammatory drugs (NSAIDs). However, the discovery of the second isozyme, PGH synthase-2 (PGHS-2), and its association with inflammation has suggested that this latter enzyme may be the therapeutic target of NSAIDs functioning in their antiinflammatory capacities. We have cloned cDNAs for murine PGHS-1 and PGHS-2, expressed these enzymes in cos-1 cells, and compared the relative sensitivities of the two isozymes to some common NSAIDs. Indomethacin, piroxicam, and sulindac sulfide were found to preferentially inhibit PGHS-1. Ibuprofen and meclofenamate inhibit both enzymes with comparable potencies. 6-Methoxy-2-naphthylacetic acid, the active metabolite of Relafen, inhibits murine PGHS-2 preferentially. Aspirin irreversibly inhibits PGHS-1, preventing this isozyme from forming PGH2 or any other oxygenated product; in contrast, aspirin treatment of PGHS-2 causes this enzyme to form 15-hydroxy-5c,8c,11c,13t-eicosatetraenoic acid (15-HETE) instead of PGH2. Our results indicate mouse PGHS-1 and PGHS-2 are pharmacologically distinct. Thus, it should be possible to develop agents highly selective for each PGHS isozyme. PGHS-2 is not expressed in stomach but is increased by inflammatory cytokines in cells such as macrophages. Thus, a selective inhibitor of PGHS-2 could be an antiinflammatory agent but without being ulcerogenic.

Animals↗
PubMed1994

Characterization of a tyrosyl radical in prostaglandin endoperoxide synthase-2.

Two different isoforms of prostaglandin H synthase, prostaglandin H synthase-1 and prostaglandin H synthase-2, have been identified. Both isozymes catalyze both cyclooxygenase and peroxidase reactions. Residues identified as being essential for catalysis by ovine prostaglandin endoperoxide H synthase-1 are all conserved in prostaglandin H synthase-2. This suggests that the enzymic reaction mechanisms are fundamentally the same for both isozymes. A tyrosyl radical, which may initiate the cyclooxygenase reaction, is detected by electron paramagnetic resonance spectroscopy after addition of arachidonic acid or hydroperoxides to ovine prostaglandin H synthase-1. We report here that human prostaglandin H synthase-2 also generates a tyrosyl radical centered at g = 2.0040 with a width of 29 gauss, similar to prostaglandin H synthase-1. This is the first spectral evidence that the two isoforms are similar mechanistically.

Animals↗
PubMed1994

The orientation of prostaglandin endoperoxide synthases-1 and -2 in the endoplasmic reticulum.

Prostaglandin endoperoxide H synthases (PGHS)-1 and -2 are integral membrane proteins of the endoplasmic reticulum (ER). The luminal versus cytoplasmic orientations of several epitopes of PGHS-1 and PGHS-2 were determined by immunocytofluorescent staining of cells following treatment with membrane-selective permeants. With serum-stimulated, murine NIH/3T3 cells expressing PGHS-2, an anti-peptide antibody directed against a domain near the COOH terminus of this isozyme caused staining only after all membranes were permeabilized with 0.2% saponin; no staining occurred with 3T3 cells treated with digitonin to permeabilize only the plasma membrane. Similarly, cos-1 cells expressing ovine PGHS-1 were stained with anti-peptide antibodies directed against (a) the amino terminus (residues 25-35), (b) a domain containing the tryptic cleavage site at Arg277 (residues 272-284), or (c) a region near the carboxyl terminus (residues 583-594) following permeabilization with saponin but not with digitonin or streptolysin O. The results obtained with the antibodies against the Arg277-containing domain of PGHS-1 were surprising because the enzyme is susceptible to tryptic cleavage at Arg277 in microsomal preparations. However, enzymatic and immunochemical analyses of microsomes prepared from ovine vesicular glands and cos-1 cells indicated that these microsomes are not intact. Accordingly, our results indicate that the trypsin cleavage site (Arg277) as well as the NH2 and COOH termini of ovine PGHS-1 are on the luminal side of the ER. The NH2 terminus, the Arg277 domain, and the N-glycosylation sites of ovine PGHS-1 are part of a large soluble, globular structure in crystalline ovine PGHS-1 (Picot, D., Loll, P. J., and Garavito, M. (1994) Nature, 367, 243-249). We conclude that PGHS-1 and, by analogy, the highly homologous PGHS-2 are luminal ER proteins. Assuming that the PGHS-1 and PGHS-2 present in the ER are functional in intact cells, our results indicate that PGH2 synthesis from arachidonate occurs in the lumen of the ER.

3T3 Cells↗
PubMed1994

Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin.

Aspirin (acetylsalicylate) treatment of human (h) prostaglandin endoperoxide H synthase (PGHS)-1 expressed in cos-1 cells caused a time-dependent inactivation of oxygenase activity. Aspirin treatment of hPGHS-2 produced an enzyme which retained oxygenase activity but formed exclusively 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) instead of PGH2. The 15-HETE was exclusively of the 15R configuration. The Km values for arachidonate of native and aspirin-treated hPGHS-2 were about the same suggesting that arachidonate binds to both aspirin-treated and native hPGHS-2 in a similar manner. If, as expected, the formation of 15R-HETE proceeds through abstraction of the 13proS hydrogen from arachidonate, O2 insertion must occur from the same side as the hydrogen abstraction; with all other lipoxygenases and cyclooxygenases, O2 addition is antarafacial. When microsomal hPGHS-2 was incubated with [acetyl-14C]aspirin, the enzyme was acetylated. An S516A mutant of hPGHS-2, which retains enzyme activity, was not acetylated. This indicates that Ser-516 is the site of aspirin acetylation of hPGHS-2; this residue is homologous to the "active site" serine of PGHS-1. An S516N mutant of hPGHS-2 was catalytically active; in contrast, an S516Q mutant lacked cyclooxygenase but retained peroxidase activity. Because in the case of PGHS-1 a smaller asparagine substitution is sufficient to eliminate cyclooxygenase activity, we conclude that the active site of PGHS-2 is slightly larger than that of PGHS-1. An S516M mutant of hPGHS-2 was obtained which resembled aspirin-acetylated hPGHS-2 in that this mutant made 15R-HETE as its major product; however, unlike the aspirin-acetylated hPGHS-2, the Km value of the S516M mutant for arachidonate was 100 times that of native hPGHS-2.

Acetylation↗
PubMed1994

Pharmacology of prostaglandin endoperoxide synthase isozymes-1 and -2.

It is now that there are two isozymes of prostaglandin endoperoxide (PGH) synthase (cyclooxygenase) called PGH synthase-1 and -2 or COX I and II. Both isozymes catalyze the conversion of arachidonate to PGH2, the committed step in the formation of both prostacyclin and thromboxane A2. PGH synthase-1 is present in platelets and endothelial cells whereas PGH synthase-2 has been detected in endothelial cells treated with cytokines and phorbol esters. PGH synthase-1 (PGHS-1) has long been thought to be the site of action of nonsteroidal anti-inflammatory drugs (NSAIDs). However, it is now clear that the second isozyme, PGH synthase-2 (PGHS-2), is also inhibited by these compounds. Cloning of the cDNAs for murine PGHS-1 and PGHS-2 has allowed us to express these two enzymes in cos-1 cells and to compare the relative sensitivities of these enzymes in vitro using a series of common NSAIDs. NSAIDs such as indomethacin, piroxicam, and sulindac sulfide preferentially inhibit PGHS-1. Ibuprofen and meclofenamate inhibit both enzymes with comparable potencies. 6-Methoxy-2-naphthylacetic acid, the active metabolite of Relafen, inhibits murine PGHS-2 preferentially. Aspirin irreversibly inhibits PGHS-1, preventing this isozyme from forming any product; in contrast, aspirin treatment of PGHS-2 causes this enzyme to form 15-hydroxy-5c,8c,11c,13t-eicosatetraenoic acid (15-HETE) instead of PGH2. These results establish that the two mouse enzymes are pharmacologically distinct and suggest that it will be possible to identify or design compounds that are completely selective for each PGHS isozyme. Because PGHS-2 is usually only expressed in inflamed tissue or after exposure to mediators of inflammation, a selective inhibitor of this isoenzyme may exhibit anti-inflammatory activity without effects on PGHS-1 of platelets and perhaps without ulcerogenic effects associated with commonly available NSAIDs.

Animals↗
PubMed1994

Characterization of the tyrosyl radicals in ovine prostaglandin H synthase-1 by isotope replacement and site-directed mutagenesis.

Several free radical species, attributed to tyrosyl radicals, have been previously observed by electron paramagnetic resonance (EPR) spectroscopy during reaction of pure ovine prostaglandin H synthase-1 (PGHS-1) with hydroperoxide or fatty acid substrates. The identity and location of amino acid residue(s) involved in formation of these radicals has been investigated using wild type and mutant PGHS-1 expressed in transfected COS-1 cells. Upon reaction with hydroperoxide, detergent extracts from microsomes of cells expressing wild type ovine PGHS-1 rapidly produced EPR signals made up of a broad component, resembling either the wide doublet or the wide singlet seen in reactions of the purified ovine enzyme, mixed with a variable proportion of a narrow component. Isotope replacement with perdeuterated tyrosine and phenylalanine in wild type PGHS-1 caused no change in enzymatic activity or capability to form a peroxide-induced radical, but the radical was an isotropic singlet without any wide features. This loss of splitting in the deuterated enzyme establishes that the wide radical signal in PGHS-1 is indeed a tyrosyl radical, validating the earlier assignment based on spectral similarities with a tyrosyl radical in ribonucleotide reductase. A Y385F mutation abolished cyclooxygenase but not peroxidase activity, and the peroxide-induced EPR was narrowed to a singlet essentially identical with that obtained with indomethacin-treated ovine enzyme. Isotopic replacement with perdeuterated tyrosine and phenylalanine in the Y385F mutant led to further narrowing of the peroxide-induced EPR to an isotropic singlet, establishing that this radical also involved a tyrosine residue. A Y355F mutant exhibited some decrease in cyclooxygenase activity, but the peroxidase activity and the peroxide-induced radical characteristics were not significantly different from those of the wild type enzyme, indicating that Tyr355 is not involved in tyrosyl radical formation in PGHS-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Imaging evaluation of artificial nipples during bottle feeding.

OBJECTIVES: To determine whether real-time ultrasonography can be used to directly visualize artificial nipples in vivo while an infant is sucking, to compare deformation differences of the artificial nipple with the human nipple during sucking, and to compare the suck mechanism used by the infant with four types of artificial nipples. DESIGN: Nonrandomized clinical study with a control group. SETTING: University-affiliated teaching hospital in Iowa City, Iowa. PARTICIPANTS: A volunteer sample of 35 healthy infants 6 to 12 weeks of age. INTERVENTION: None. MEASUREMENTS/MAIN RESULTS: Images produced by real-time ultrasound of infants during sucking using artificial nipples were measured to determine the percentage lengthening, the percentage lateral compression, and the percentage flattening of nipples. These results were compared with data obtained from studies using breast-fed infants. None of the artificial nipples lengthened like the human nipple. One artificial nipple was significantly more compressible than the human nipple and the remaining three artificial nipples. CONCLUSION: Real-time ultrasonography can be used to visualize artificial nipples in vivo during sucking.

Bottle Feeding↗
PubMed1994

In vitro antimicrobial activity of Shiva-11 against ocular pathogens.

Cecropins are antimicrobial peptides (30-35 amino acids) isolated from the hemolymph of the cecropia moth. Previous studies have demonstrated their antimicrobial efficacy against a variety of pathogens, including both gram-positive and -negative bacteria, fungi, protozoa, and enveloped viruses. To assess their therapeutic potential against ocular pathogens, we analyzed the in vitro antimicrobial activity of a synthetic cecropin analog, Shiva-11, against virulent microbial strains (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and Candida albicans). Bacterial isolates were obtained from human cases of severe ulcerative keratitis. Shiva-11 was tested at varying concentrations in bacterial suspensions containing 1-2 x 10(6) CFU/ml at 37 degrees C. Samples were plated on nutrient agar and colonies counted after 24-48 h of incubation. Shiva-11 yielded > 3 log killing of all isolates after 60 min of exposure to this compound. The results of this study indicate that Shiva-11 possesses broad-spectrum in vitro antimicrobial activity against human clinical ocular pathogens.

Animals↗
PubMed1994

The role of previous radiographs and reports in the interpretation of current radiographs.

RATIONALE AND OBJECTIVES: The authors studied radiologists' patterns of using old reports and previous radiographs in the interpretation of current radiographic studies. Supplying information from previous studies is considered necessary for accurate interpretation; however, it is not clear how this information is used. METHODS: Eighteen radiologists interpreted 305 plain-film studies in a general radiologic interpretation area. Radiologists described their perceptions of the use of previous radiographs and reports in film interpretation. RESULTS: Overall, old films were used for 99% of interpretations and previous reports were used for 65%. Prior information increased confidence in 89% of cases, influenced diagnosis in 56% of cases, and assisted in detecting new pathology in 6% of interpretations. Old radiographs were judged most valuable in documenting the progression, regression, or stability of disease in 89% of cases. Previous reports were principally used to obtain patient history (43%). In no cases did the radiologist perceive that the old reports assisted in locating new pathology. CONCLUSIONS: Old films are judged more valuable than reports for documenting disease progress, and radiologists are unlikely to accept written reports as a substitute. Previous reports are used principally to improve clinical histories, a function that might be eliminated if adequate clinical data could be captured by other means.

Attitude of Health Personnel↗
PubMed1994

Calculating the costs of undergraduate medical education in radiology.

RATIONALE AND OBJECTIVES: The current stringent financial constraints mandate that radiology departments carefully assess the costs involved in undergraduate medical education. We performed a cost-accounting analysis of the radiology department's participation in an introductory clinical medicine course, which could serve as a methodologic model for realistic cost assessment of any new or existing course. MATERIALS AND METHODS: Each day of the 5-week radiology portion of the second-year course consists of 1 hour of lecture and 5 hours of small-group discussions regarding chest, abdominal, and skeletal radiology; neuroradiology; and nuclear medicine. Marginal cost is defined as the additional real cost involved in creating or conducting a course. Opportunity costs represent alternative use of instructional resources. RESULTS: The marginal cost (additional real cost) of providing the course for the first time was $37,475. Subsequent offerings of the course will cost approximately $24,375. CONCLUSION: Resources expended for any instructional purpose must be weighed in terms of marginal and opportunity costs. It is mandatory that adequate cost analysis for undergraduate education in radiology be developed so that sound choices can be made regarding resource consumption.

Costs and Cost Analysis↗
PubMed1994

Plasma levels of peptide YY correlate with cisplatin-induced emesis in dogs.

The effect of cisplatin on plasma peptide YY (PYY) and 5-hydroxytryptamine (5-HT) concentrations was determined in conscious dogs (n = 6 per group) pretreated with either saline, or the 5-HT3-receptor antagonists ondansetron or granisetron. Cisplatin (3.0 mg kg-1, i.v.) caused emesis (18.8 +/- 2.9 episodes; 75-284 min) and significantly increased the mean area under the curve (AUC) over a 6-h period of plasma PYY concentrations (7.4 +/- 1.8 to 11.5 +/- 3.7 ng) in all saline-pretreated dogs, whereas the mean AUC of plasma 5-HT concentrations did not significantly increase (34.7 +/- 7.4 vs 35.6 +/- 12.3 pM h). The concentrations of PYY correlated closely with the incidence of emesis (r = 0.99). In animals pretreated (36 min) with ondansetron (0.316 mg kg-1, i.v.) or granisetron (0.316 mg kg-1, i.v.), the number of cisplatin-induced emetic episodes was significantly (P < 0.005) decreased compared with control. In animals receiving cisplatin and pretreated with ondansetron, PYY concentrations were not significantly altered, whereas the mean AUC of plasma concentrations of 5-HT over 6 h increased (35.6 +/- 12.3 to 82.3 +/- 34.6 pM h; P < 0.05). In animals receiving cisplatin and pretreated with granisetron, plasma concentrations of 5-HT were not significantly altered, whereas the mean AUC of plasma PYY concentrations were significantly reduced compared with control (6.2 +/- 1.7 vs 11.5 +/- 3.7 ng h).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Factors affecting the future numbers of diagnostic radiologists.

There is a widespread conviction among health policy experts that a severe imbalance exists in the ratio of primary care to specialist physicians. There are too many specialists, including radiologists, and several health manpower planning commissions have recommended steps to change the "imbalance". The federal government, because of its position in residency funding, proposes to play a major role in the implementation of the changes in the number of medical specialists. In contrast to the discord surrounding other aspects of medical reform, the major agencies have considerable accord on the need for reducing the supply of specialists and are close on the method for doing so. This article reviews the current situation, outlines the proposals for action, and speculates on likely scenarios for their implementation.

Financing, Government↗
PubMed1994

Differential inhibition of human prostaglandin endoperoxide H synthases-1 and -2 by nonsteroidal anti-inflammatory drugs.

We developed an in vitro expression system for accurate kinetic analyses of the inhibition of the human prostaglandin H synthase isozymes (hPGHS-1 and -2) by nonsteroidal anti-inflammatory drugs (NSAIDs). Assays of instantaneous inhibition in which enzyme, 10 microM arachidonate, and an NSAID were mixed simultaneously were used to determine apparent affinities of 14 common NSAIDs for hPGHS-1 and hPGHS-2. All NSAIDs except salicylate had appreciable apparent affinities (IC50 < or = 100 microM) for hPGHS-1. Most NSAIDs also exhibited appreciable affinities toward hPGHS-2, but three prominent exceptions were indomethacin, piroxicam and phenylbutazone. We subsequently performed measurements of time-dependent inhibition in which either (a) enzyme and an NSAID were preincubated before substrate was added to initiate the reactions or (b) recovery of activity after time-dependent inhibition was measured using intact cells preincubated with various NSAIDs. Indomethacin, flurbiprofen, meclofenamate and diclofenac, but not ibuprofen, piroxicam or phenylbutazone, caused time-dependent inhibition of both hPGHS-1 and -2 in vitro. For cells pretreated with flurbiprofen or meclofenamate, hPGHS-2 activities, but not hPGHS-1 activities, were recovered relatively rapidly; with indomethacin, recoveries of hPGHS-1 and hPGHS-2 activities were both slow. hPGHS-2 is thought to be the target of NSAIDs acting as anti-inflammatory agents. However, our results indicate that neither measurements of affinities of NSAIDs for hPGHS-2 conducted in vitro with 10 microM arachidonate nor measurements of time-dependent inhibition of hPGHS-2 always predict whether a compound (e.g., piroxicam or phenylbutazone) has anti-inflammatory activity in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1993

Lethal bone dysplasia in a fetus with manifestations of atelosteogenesis I and Boomerang dysplasia.

Atelosteogenesis I (AT-I) and Boomerang dysplasia have been described as separate lethal bone dysplasias. The possibility of a common cause of both conditions was suggested by Hunter and Carpenter (Clin Genet 39(6): 471-480, 1991) in their report of a patient with apparent manifestations of both AT-I and Boomerang dysplasia. We report on a male fetus of 31 weeks gestation whose clinical, radiologic and histologic findings are compared to reported cases of AT-I, Boomerang dysplasia and the patient of Hunter and Carpenter (Clin Genet 39(6): 471-480, 1991). From the documentation of clinical and radiologic findings we demonstrate overlap of AT-I and Boomerang dysplasia in our patient, and, from histologic examination, suggest a defect of cartilage and bone formation as the basic abnormality in this lethal bone dysplasia.

Bone Diseases, Developmental↗
PubMed1993

N-glycosylation of prostaglandin endoperoxide synthases-1 and -2 and their orientations in the endoplasmic reticulum.

Using site-directed mutagenesis, we have determined that Asn68, Asn144, and Asn410 of ovine prostaglandin endoperoxide (PGH) synthase-1 are N-glycosylated. A fourth consensus N-glycosylation sequence at Asn104 is not glycosylated. Glycosylation of PGH synthase-1 at Asn410 and at either Asn68 or Asn144 was required for expression of both the cyclooxygenase and the peroxidase activities of the enzyme. Inactive PGH synthase-1 glycosylation site mutant proteins do not appear to achieve their native conformations. However, the native enzyme, once in an active conformation, does not appear to require attached carbohydrate for cyclooxygenase or peroxidase activities. N-Glycosylation consensus sequences corresponding to the three glycosylation sites of ovine PGH synthase-1 are conserved in the deduced amino acid sequences of PGH synthases-2. Using site-directed mutagenesis, we determined that there is an additional site of N-glycosylation in murine PGH synthase-2 located at Asn580. This site is N-glycosylated in about 50% of PGH synthase-2 molecules, resulting in two peptide bands on SDS-polyacrylamide gel electrophoresis (72 and 74 kDa). Glycosylation of PGH synthase-2 is necessary for expression of enzyme activity, but glycosylation of PGH synthase-2 at Asn580 per se does not affect activity. Assuming that the N-glycosylation sites of PGH synthase-1 are on the luminal side of the endoplasmic reticulum (ER), and that the site of tryptic cleavage of ovine PGH synthase-1 (Arg277) is on the cytoplasmic side of the ER, we propose that both the NH2 and COOH termini of PGH synthase-1 are located in the lumen of the ER and that there are two transmembrane domains located between Asn144 and Arg277 and between Arg277 and Asn410, respectively. A similar orientation is predicted for PGH synthase-2.

Amino Acid Sequence↗