Search PubMed⌕ Search

Biomedical subjects

W L Smith

Publications and source records attributed to W L Smith.

At least 19 recordsLinked to original sources
PubMed1996

C-terminal Ser/Pro-Thr-Glu-Leu tetrapeptides of prostaglandin endoperoxide H synthases-1 and -2 target the enzymes to the endoplasmic reticulum.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) are integral membrane proteins associated with the luminal surface of the endoplasmic reticulum (ER) and the nuclear envelope (NE). The C-terminal sequences of PGHSs end in -Ser/Pro-Thr-Glu-Leu (-S/PTEL), a sequence similar to one of the known ER targeting sequences, -Lys-Asp-Glu-Leu (-KDEL). Previous immunofluorescence studies from our own and another laboratory had indicated that both native ovine (o) PGHS-1 and oPGHS-1 mutants with modified or deleted -PTEL sequences were localized primarily in the ER when the proteins were expressed for 24-40 h following transient transfection of cos-1 cells. However, in characterizing an oPGHS-1 mutant lacking 15 amino acids from the C-terminus (deltaCT15 oPGHS-1), we found that when this mutant was expressed for a shorter period (18 h) following transfection, the enzyme was concentrated near the nucleus in what appeared to be the Golgi apparatus; similar results were observed when the -P/STEL mutants of oPGHS-1 prepared previously (i.e., deltaPTEL, L600N, and L600R oPGHS-1) were retested using the 18-h posttransfection expression time; in contrast, under the same conditions, the native enzyme was localized throughout the cytoplasm (i.e., in the ER). When the cos-1 cells transfected with each of the various C-terminal mutants were treated with Brefeldin A, the immunofluorescent staining was redistributed to the ER, whereas the distribution of native oPGHS-1 was unaffected. A human PGHS-2 (hPGHS-2) mutant in which the C-terminal leucine residue was replaced with an asparagine (hPGHS-2 L604N) was also found to localize to the Golgi apparatus following 18 h expression in transfected cos-1 cells. These results establish that the C-terminal-S/PTEL sequences of both PGHS-1 and PGHS-2 target the enzymes to the ER. PGHS-2 is more highly concentrated in the NE than in the ER, but apparently this isozyme traverses the Golgi apparatus and returns to the ER prior to its becoming concentrated in the NE.

Amino Acid Sequence↗
PubMed1996

Regulation of prostaglandin endoperoxide H synthase-2 expression by 2,3,7,8,-tetrachlorodibenzo-p-dioxin.

We have examined the molecular mechanisms for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-stimulated prostaglandin synthesis in Mardin Darvey canine kidney cells (MDCK). TCDD stimulates prostaglandin synthesis in these cells, at least in part, by elevating prostaglandin endoperoxide H2 synthase-2 (PGHS-2) levels. TCDD-stimulated transcription of the PGHS-2 gene was maximal (6-fold) within 2 h and resulted in a 100-fold increase in PGHS-2 mRNA and a 25-fold increase in PGHS-2 protein levels by 4 h. Transient transfection experiments using luciferase-reporter plasmids demonstrated that control element(s) responsible for TCDD activation of the murine PGHS-2 promoter in MDCK cells are located in the first 965 nucleotides upstream from the PGHS-2 transcriptional initiation site. A canonical xenobiotic response element, similar to those that control transcription of other well-known TCDD-sensitive genes, is present at position -157, but does not appear to be sufficient for halogenated aromatic hydrocarbon (HAH) activation of the PGHS-2 promoter. TCDD failed to stimulate transcription from the PGHS-2 promoter when reporter plasmids were transfected into Hepa 1c1c7 cells, a line which contains the functional aryl hydrocarbon receptor. It seems likely that inappropriate expression of PGHS-2 may contribute to the toxic effects of TCDD and other HAHs. In particular, PGHS-2 expression may affect those toxic reactions that involve inappropriate cellular growth, such as dermal hyperplasia and tumor formation. It is also likely that elevated synthesis of prostaglandins, which are potent regulators of immune function, could play a role in the immunotoxicity associated with HAH exposure.

Amino Acid Sequence↗
PubMed1996

Photolabeling of prostaglandin endoperoxide H synthase-1 with 3-trifluoro-3-(m-[125I]iodophenyl)diazirine as a probe of membrane association and the cyclooxygenase active site.

Previous studies of the crystal structure of the ovine prostaglandin endoperoxide H synthase-1 (PGHS-1)/S-flurbiprofen complex (Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249) suggest that the enzyme is associated with membranes through a series of four amphipathic helices located between residues 70 and 117. We have used the photoactivatable, hydrophobic reagent 3-trifluoro-3-(m-[125I]iodophenyl)diazirine ([125I]TID) which partitions into membranes and other hydrophobic domains to determine which domains of microsomal PGHS-1 are subject to photolabeling. After incubation of ovine vesicular gland microsomes with [125I]TID, ovine PGHS-1 was one of the major photolabeled proteins. Proteolytic cleavage of labeled PGHS-1 at Arg277 with trypsin established that [125I]TID was incorporated into both the 33-kDa tryptic peptide containing the amino terminus and the 38-kDa tryptic peptide containing the carboxyl terminus. This pattern of photolabeling was not affected by the presence of 20 mM glutathione, indicating that the photolabeling observed for PGHS-1 was not due to the presence of [125I]TID in the aqueous phase. However, nonradioactive TID as well as two inhibitors, ibuprofen and sulindac sulfide, which bind the cyclooxygenase active site of PGHS-1, prevented the labeling of the 38-kDa carboxyl-terminal tryptic peptide. These results suggest that [125I]TID can label both the cyclooxygenase active site in the tryptic 38-kDa fragment and a membrane binding domain located in the 33-kDa fragment. Cleavage of photolabeled PGHS-1 with endoproteinase Lys-C yielded a peptide containing residues 25-166 which was labeled with [125I]TID. This peptide contains the putative membrane binding domain of ovine PGHS-1. Our results provide biochemical support for the concept developed from the crystal structure that PGHS-1 binds to membranes via four amphipathic helices located near the NH2 terminus of the protein.

Affinity Labels↗
PubMed1996

Involvement of arginine 120, glutamate 524, and tyrosine 355 in the binding of arachidonate and 2-phenylpropionic acid inhibitors to the cyclooxygenase active site of ovine prostaglandin endoperoxide H synthase-1.

Examination of the crystal structure of the ovine prostaglandin endoperoxide synthase-1 (PGHS-1)/S- flurbiprofen complex (Picot, D., Loll, P.J., and Garavito, R.M. (1994) Nature 367, 243-2491) suggests (a) that the carboxyl group of arachidonic acid interacts with the arginino group of Arg120; (b) that Arg120 forms an important salt bridge with Glu524; and (c) that Tyr355, which is in close proximity to Arg120, could determine the stereochemical specificity of PGHS-1 toward 2-phenylpropionic acid inhibitors. To test these concepts, we used site-directed mutagenesis to prepare ovine PGHS-1 mutants having modifications of Arg120 (R120K, R120Q, R120E), Glu524 (E524D, E524Q, E524K), and Tyr355 (Y355F) and examined the properties of the mutant enzymes expressed in COS-1 cells. All of the mutants retained at least part of their cyclooxygenase and peroxidase activities except the R120E mutant, which had no detectable activity. The Km values of the R120K and R120Q mutants with arachidonic acid were 87 and 3300 microM, respectively, versus 4 microM for native PGHS-1. The R120Q mutant failed to undergo suicide inactivation during catalysis or time-dependent inhibition by flurbiprofen. These results are consistent with Arg120 binding the carboxylate group of arachidonate and suggest that interaction of the carboxylate group of substrates and inhibitors with Arg120 is necessary for suicide inactivation and time-dependent inhibition, respectively. The Km values for the E524D, E524Q, and E524K mutants were not significantly different from values obtained for the native PGHS-1, suggesting that this residue is not importantly involved in catalysis or substrate binding. The effect of modifications of Arg120 and Tyr355 on the stereospecificity of inhibitor binding was determined. Ratios of IC50 values for cyclooxygenase inhibition by D- and L-ibuprofen, a competitive cyclooxygenase inhibitor, were 32, 67, and 7.1 for native PGHS-1, R120Q PGHS-1, and Y355F PGHS-1, respectively. The decreased stereochemical specificity observed with the Y355F PGHS-1 mutant suggests that Tyr355 is a determinant of the stereospecificity of PGHS-1 toward inhibitors of the 2-phenylpropionic acid class.

Amino Acid Sequence↗
PubMed1996

A Dejerine-Sottas neuropathy family with a gene mapped on chromosome 8.

The Patient is a 55-year-old black male who belongs to a large family with 9 affected relatives with autosomal dominant Dejerine-Sottas neuropathy (DSN). Onset of his condition was at 2 years of age with steppage gait followed by severe progressive weakness, atrophy, and sensory loss of his legs and hands accompanied by areflexia and thoracolumbar kyphoscoliosis. The patient became wheelchair confined at age 38. At around age 42, the left shoulder became dislocated and the humeral head underwent aseptic necrosis (Charcot joint). Nerve conduction studies showed absent motor and sensory responses for all major nerves tested. Genetic linkage suggested mapping of this DSN gene on chromosome 8qter. A younger brother with similar neurological findings also demonstrated Charcot joints with bone destruction of the joints of the fourth and fifth fingers.

Chromosome Mapping↗
PubMed1996

Involvement of prostaglandin endoperoxide H synthase-2 in osteoclast-like cell formation induced by interleukin-1 beta.

Interleukin-1 beta (IL-1 beta) stimulates osteoclast-like cell formation via prostaglandin E2 (PGE2) production. However, the regulatory mechanism for the production of PGE2 in bone cells is still unclear. Recently, it has been shown that two prostaglandin endoperoxide H synthase (PGHS) isozymes exist, termed PGSH-1 and PGHS-2. We report here that IL-1 beta induces PGE2 production in bone marrow culture induced by a PGHS-2-dependent mechanism. IL-1 beta stimulated the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC) and the production of PGE2 in mouse bone marrow cultures. The dose response curves for the indomethacin inhibition of TRAP-positive MNC formation and PGE2 production were nearly identical. Cycloheximide (CHX) suppressed IL-1 beta-induced PGE2 production, suggesting that the production of PGE2 induced by IL-1 beta required de novo protein synthesis. Northern blot analysis determined that IL-1 beta induced PGHS-2 expression by 30 minutes and mRNA levels were maximal by 1-2 h. Cycloheximide potentiated the accumulation of PGHS-2 mRNA linearly up to 8 h. Dexamethasone, an inhibitor of the induction of PGHS-2, inhibited IL-1 beta-induced PGHS-2 mRNA expression and also suppressed IL-1 beta-stimulated formation of TRAP-positive MNC. Furthermore NS-398, as a selective PGHS-2 inhibitor, completely inhibited IL-1 beta-induced TRAP-positive MNC formation. Moreover, IL-1 beta-induced PGHS-2 mRNA expression and formation of TRAP-positive MNC were inhibited by calphostin C, a selective inhibitor of protein kinase C (PKC). These results indicate that IL-1 beta-induced formation of osteoclast-like cells requires PKC activation, induction of PGHS-2, and subsequent prostaglandin synthesis by this enzyme.

Animals↗
PubMed1996

Civility.

Explore the source record for details and available documents.

Ethics, Medical↗
PubMed1996

Philanthropy.

Explore the source record for details and available documents.

Fund Raising↗
PubMed1996

Duplex Doppler sonography of changes in portal vein flow in healthy term newborn infants after feeding.

To establish reference values for changes in portal venous diameter, angle- corrected maximal flow velocity, and flow in healthy term newborn infants after formula feeding, we studied 20 subjects using duplex Doppler sonography. After feeding, portal venous diameter increased from 3.6 +/- 0.1 (mean +/- standard error of the mean) to 3.9 +/- 0.1 mm at 15 min and decreased to 3.8 +/- 0.1 at 60 min. Maximal flow velocity increased from 24.1 +/- 1.3 cm/s to a maximum of 35.9 +/- 2.4 cm/s at 15 min and decreased to 28.8 +/- 1.5 cm/s at 60 min. Flow increased from 85.0 +/- 7.5 ml/min to a maximum of 153.6 +/- 14.9 ml/min at 15 min with decrease similar to the maximal flow velocity curve. We conclude that formula feeding produces peak portal blood flows of nearly twice the fasting values at 15 min after feeding and returns almost to fasting value by 60 min.

Eating↗
PubMed1996

Inflammatory bowel disease in children.

Pediatric inflammatory bowel disease is a complex area of pediatric gastrointestinal imaging. Multiple causative factors are discussed with emphasis upon disorders without known or presumed infectious etiology. Conditions discussed include Henoch-Schönlein purpura, Kawasaki disease, Behçet's disease, Crohn's disease, and ulcerative colitis with their respective clinical and radiological findings, differential diagnosis features, plus pertinent extraintestinal manifestations of inflammatory bowel disease.

Child↗
PubMed1995

Calcium-mediated translocation of cytosolic phospholipase A2 to the nuclear envelope and endoplasmic reticulum.

Cytosolic phospholipase A2 (cPLA2) is activated by a wide variety of stimuli to release arachidonic acid, the precursor of the potent inflammatory mediators prostaglandin and leukotriene. Specifically, cPLA2 releases arachidonic acid in response to agents that increase intracellular Ca2+. In vitro data have suggested that these agents induce a translocation of cPLA2 from the cytosol to the cell membrane, where its substrate is localized. Here, we use immunofluorescence to visualize the translocation of cPLA2 to distinct cellular membranes. In Chinese hamster ovary cells that stably overexpress cPLA2, this enzyme translocates to the nuclear envelope upon stimulation with the calcium ionophore A23187. The pattern of staining observed in the cytoplasm suggests that cPLA2 also translocates to the endoplasmic reticulum. We find no evidence for cPLA2 localization to the plasma membrane. Translocation of cPLA2 is dependent on the calcium-dependent phospholipid binding domain, as a calcium-dependent phospholipid binding deletion mutant of cPLA2 (delta CII) fails to translocate in response to Ca2+. In contrast, cPLA2 mutated at Ser-505, the site of mitogen-activated protein kinase phosphorylation, translocates normally. This observation, combined with the observed phosphorylation of delta CII, establishes that translocation and phosphorylation function independently to regulate cPLA2. The effect of these mutations on cPLA2 translocation was confirmed by subcellular fractionation. Each of these mutations abolished the ability of cPLA2 to release arachidonic acid, establishing that cPLA2-mediated arachidonic acid release is strongly dependent on both phosphorylation and translocation. These data help to clarify the mechanisms by which cPLA2 is regulated in intact cells and establish the nuclear envelope and endoplasmic reticulum as primary sites for the liberation of arachidonic acid in the cell.

Animals↗
PubMed1995

Fatty acid substrate specificities of human prostaglandin-endoperoxide H synthase-1 and -2. Formation of 12-hydroxy-(9Z, 13E/Z, 15Z)- octadecatrienoic acids from alpha-linolenic acid.

Human prostaglandin-endoperoxide H synthase-1 and -2 (hPGHS-1 and hPGHS-2) were expressed by transient transfection of COS-1 cells. Microsomes prepared from the transfected cells were used to measure the rates of oxygenation of several 18- and 20-carbon polyunsaturated fatty acid substrates including eicosapentaenoic, arachidonic, dihomo-gamma-linolenic > alpha-linolenic (delta 9, 12, 15), gamma-linolenic, and linoleic acids. Comparisons of kcat/Km values indicate that the order of efficiency of oxygenation is arachidonate > dihomo-gamma-linolenate > linoleate > alpha-linolenate for both isozymes; while the order of efficiency was the same for hPGHS-1 and hPGHS-2, alpha-linolenate was a particularly poor substrate for hPGHS-1. Gamma-Linolenate and eicosapentaenoate were poor substrates for both isozymes, but in each case, these two fatty acids were better substrates for hPGHS-2 than hPGHS-1. These studies of substrate specificities are consistent with previous studies of the interactions of PGHS isozymes with nonsteroidal anti-inflammatory drugs that have indicated that the cyclooxygenase active site of PGHS-2 is somewhat larger and more accommodating than that of PGHS-1. The major products formed from linoleate and alpha-linolenate were characterized. 13-Hydroxy-(9Z,11E)-octadecadienoic acid was found to be the main product formed from alpha-linoleate by both isozymes. The major products of oxygenation of alpha-linolenate were determined by mass spectrometry to be 12-hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic acids. This result suggests that alpha-linolenate is positioned in the cyclooxygenase active site with a kink in the carbon chain such that hydrogen abstraction occurs from the omega 5-position in contrast to abstraction of the omega 8-hydrogen from other substrates.

Animals↗
PubMed1995

The virtual hospital. Providing multimedia decision support tools via the Internet.

Physicians in the clinical setting remain isolated from important sources of medical information. The authors have created a multimedia database known as The Virtual Hospital that improves access to current medical data, which is used to improve patient care decisions. The Virtual Hospital is a digital health sciences library stored on a server (computer) at The University of Iowa and delivered via the Internet to inexpensive personal computers in the workplace. The emerging standard of the World Wide Web is used to provide cross-platform distribution.

Computer Communication Networks↗
PubMed1995

Expression-activity profiles of cells transfected with prostaglandin endoperoxide H synthase measured by quantitative fluorescence microscopy.

Transfection of cos-1 cells with either prostaglandin endoperoxide H synthase-1 (PGHS-1) or -2 (PGHS-2) results in a mixed population of cells containing a diverse range of expressed enzyme. The use of fluorescent substrates and antibodies, in conjunction with fluorescence microscopy, provides the means to quantitate expression and activity of the enzyme within individual cells. Data obtained from individual cells can be utilized to construct enzyme activity curves for a population of transfected cells. This method has been employed to prepare expression-activity profiles within a population of cos-1 cells expressing PGHS-1 or -2. A direct correlation was observed between enzyme expression and activity as measured in single cells. The data demonstrate that activity-expression analyses can now be performed within single adherent cells growing in tissue culture.

Amino Acid Sequence↗