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Biomedical subjects

P R Young

Publications and source records attributed to P R Young.

At least 19 recordsLinked to original sources
PubMed1992

Inhibition of Clostridium perfringens phospholipase C by ammonium and sulfonium dications.

Polyamines, alkyldiammonium and alkyldisulfonium salts, inhibit the Clostridium perfringens phospholipase C-catalyzed hydrolysis of 1-S-phosphocholine-2-O-hexadecanoyl-1-mercapto-2-ethanol (1) at pH 7.5, 37 degrees C, mu = 1.0 with KCl. Simple saturation kinetics are observed as both 1 and [Ca2+] are varied and simple linear inhibition is observed. The data are consistent with a non-competitive mechanism that involves binding of the inhibitors to free enzyme, E.[Ca2+] and E.[Ca2+].[S]; the inhibition constants for decamethylenebis(trimethylammonium) and decamethylenebis(dimethylsulfonium) bromides are 90 and 0.28 mM, respectively. It is suggested that the enhanced inhibition by the alkyldisulfonium salts results from more favorable equilibrium constants for contact ion-pair formation or from the formation of tetracoordinate sulfuranes.

Ammonia↗
PubMed1992

Substratum acidification by murine B16F10 melanoma cultures.

Murine B16F10 melanoma cells, adherent to thin films of crosslinked, fluorescein-labeled collagen I, and covered by a thin layer of 0.7% agarose, exhibit a decrease in fluorescence emission in the substratum region immediately beneath adherent cells. The relative diminution in fluorescence intensity is dependent on excitation wavelength and is observed following excitation at 490 nm, but is not observed following excitation at 452 nm. The decrease in fluorescence emission is not due to quenching or concentration effects and is attributed to the decrease in extracellular pH in the substratum region. Fluorescence measurements of (I490/I452) within these substratum regions, correlate with an average extracellular pH of 6.4 +/- 0.2 which drops to pH less than 5 after 5 h. It is suggested that this region is sufficiently acidic to activate secreted or cell-surface acid proteinase enzymes and that the activity of these enzymes may be important in invasiveness by this cell-line.

Animals↗
PubMed1992

Bovine brain cathepsin D: inhibition by pepstatin and binding to concanavalin A.

1. Cathepsin D from bovine brain has been purified 1100-fold in 46% recovery. Three isozymes are present with pI (+/- 0.05) = 6.10, 6.30 and 6.40. 2. The isozymes are single polypeptide chains with apparent Mr = 42,000 and are similar with respect to substrate binding and cleavage; the pH-optimum is 3.5 with virtually no activity at neutral pH. 3. Pepstatin inhibits the enzyme and kinetic data are consistent with a "tight binding" mechanism. 4. The dissociation constant for the concanavalin A-enzyme complex is Kd = 19 nM at pH 5.0. 5. Under conditions where 90% of the enzyme is bound to soluble concanavalin A, full enzymatic activity is observed.

Animals↗
PubMed1992

Anomalies associated with myelodysplasia.

The records of 181 patients with myelodysplasia were reviewed. The prevalence of associated genitourinary anomalies as well as cardiac, facial, anal, and tethered cord were determined. The prevalence of congenital malformations associated with myelodysplasia were less in this series than in previous reports.

Abnormalities, Multiple↗
PubMed1992

The discovery and development of zileuton: an orally active 5-lipoxygenase inhibitor.

The enzyme 5-lipoxygenase is a key target in the effort to discover drugs which inhibit the pathophysiology associated with the formation of leukotrienes. The research efforts of these laboratories have focused on the discovery of direct enzyme inhibitors of 5-lipoxygenase. In particular, compounds with hydroxamate or N-hydroxyurea functionalities have proven to be potent inhibitors of leukotriene biosynthesis in vitro and more importantly in vivo. One of these compounds, zileuton (N-(1-benzo-[b]-thien-2-ylethyl)-N-hydroxyurea) has been shown recently to be an effective leukotriene inhibitor in man. The critical approaches and breakthroughs in the discovery and development of zileuton are described. In addition, some recent results with zileuton in animals and man are detailed.

Animals↗
PubMed1992

Protein hormones and their receptors.

Technological advances in the isolation and characterization of novel receptors have led to a significant increase in our understanding of protein-ligand binding to receptors and the means by which responses are triggered. Hormones and their receptors are composed of structurally conserved domains, and several ligands appear to use similar surface regions for receptor binding. A key event in signal transduction is the aggregation by the ligand of one or more receptor subunits, and this can include the sharing of subunits between different ligands. These findings have allowed the design of ligands with receptor-antagonist properties.

Hormones↗
PubMed1992

Further aspects of IL-1 beta secretion revealed by transfected monkey kidney cells.

Because the cytokine interleukin-1 beta (IL-1 beta) lacks a classical hydrophobic signal sequence, it has been unclear how it is released from cells, and whether release proceeds via a novel mechanism or through non-specific leakage. To address this issue, we have examined the secretion of the recombinant forms of human IL-1 beta from COS monkey kidney cells, which express low levels of endogenous IL-1 beta. Four proteins were expressed: precursor and mature IL-1 beta and precursor and mature IL-1 beta fused to an amino terminal hydrophobic signal sequence from human tissue plasminogen activator. By monitoring the appearance of a known cytosolic protein (ATP citrate lyase) in the medium, we find that the unmodified IL-1 beta s are non-specifically released in very small quantities from the cytosol. On the other hand, the signal sequence-modified IL-1 beta s are glycosylated and efficiently secreted by the ER/Golgi pathway. The secreted, modified-mature protein is also biologically active, suggesting that this pathway has been bypassed for reasons other than maintaining the structural integrity of IL-1 beta. More likely the alternative pathway is a critical aspect of IL-1 biology. The differences in kinetics and quantity of IL-1 beta release from monocytic and COS cells suggest that COS cells lack critical components for the rapid release seen in monocytes.

Animals↗
PubMed1991

Inhibition of leukotriene biosynthesis in the rat peritoneal cavity.

In the search for a model of leukotriene (LT) production to provide a method to determine in vivo 5-lipoxygenase (5-LO) inhibitory activity by various compounds, a passive anaphylactic reaction in the rat peritoneal cavity was examined, refined and characterized. The reaction, produced by passive sensitization with an i.p. injection of rabbit anti-bovine serum albumin (anti-BSA) followed by an i.p. injection of BSA, resulted in the biosynthesis of large amounts of sulfidopeptide LTs measurable by immunoassay or by reversed phase high performance liquid chromatography. The oral activity of several 5-LO inhibitors has been examined using this model. An example of these is zileuton (Abbott-64077), a potent 5-lipoxygenase inhibitor now under clinical evaluation. Zileuton inhibited sulfidopeptide LT biosynthesis in the rat peritoneal cavity in a dose-dependent manner (ED50 = 3 mg/kg). WY-49,232, MK-866, BW A4C and phenidone also produced good activity with ED50 values of 6, 8, 11 and 17 mg/kg, respectively. This modified rat peritoneal anaphylaxis model appears to be a valuable tool for establishing in vivo activity of 5-LO inhibitors.

Administration, Oral↗
PubMed1991

Kinetic mechanism of Clostridium perfringens phospholipase C. Hydrolysis of a thiophosphate analogue of lysophosphatidylcholine.

The hydrolysis of S-[2-(hexadecanoyloxy)ethyl]thiophosphocholine (I), an analogue of lysophosphatidylcholine, by Clostridium perfringens phospholipase C, was followed at pH 7.5, 37 degrees C and I 1.0 (maintained with KCl), in a continuous assay, by monitoring the reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) at 412 nm. Simple saturation kinetics are observed with linear mixed-type slope-intercept effects for the hydrolysis of compound (I) with variable [Ca2+] at fixed concentrations of compound (I) and a simple slope effect as [compound (I)] is varied at fixed concentrations of Ca2+. These data are consistent with a simple ordered rapid-equilibrium mechanism in which Ca2+ binds to the enzyme first followed by substrate. The observed kinetic constants at pH 7.5, 37 degrees C and I 1.0 are K1 = 12.0 mM (Ca2+ dissociation), K2 = 36 microM [compound (I) dissociation] and Vmax. = 552 microM.min-1.mg-1. Alkane diammonium salts inhibit the enzyme by a non-competitive mechanism that involves binding to free enzyme, E.Ca2+ and E.Ca2+.S. The use of the simple micellarized substrate under these conditions allows the determination of kinetic and inhibition constants without complications arising from enzyme-micelle interactions.

Clostridium perfringens↗
PubMed1991

Binding of cathepsin D to the mannose receptor on rat peritoneal macrophages.

Adherent cultures of rat peritoneal macrophages secrete lysozyme and the lysosomal marker enzymes beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase; the levels of secreted lysosomal cathepsin D, however, were found to be insignificant. Incubation of the cells at 4 degrees C for 15 min with yeast mannan or with 50 mM mannose, methyl alpha-glucopyranoside, or N-acetylglucosamine caused the concentration of cathepsin D in the culture medium to increase 30-40-fold; mannose-6-phosphate had no effect. 125I-labeled cathepsin D was prepared and the binding constant to the macrophage cell surface was determined to be KD = 27 nM. The data suggest that cathepsin D binds to the mannose receptor of macrophages and that binding to this receptor is not in equilibrium with the bulk medium.

Acetylglucosamine↗
PubMed1991

Secretion of N-glycosylated interleukin-1 beta in Saccharomyces cerevisiae using a leader peptide from Candida albicans. Effect of N-linked glycosylation on biological activity.

Human interleukin-1 beta (IL-1 beta) is expressed in activated monocytes as a 31-kDa precursor protein which is processed and secreted as a mature, unglycosylated 17-kDa carboxyl-terminal fragment, despite the fact that it contains a potential N-linked glycosylation site near the NH2 terminus (-Asn7-Cys8-Thr9-). cDNA coding for authentic mature IL-1 beta was fused to the signal sequence from the Candida albicans glucoamylase gene, two amino acids downstream from the signal processing site. Upon expression in Saccharomyces cerevisiae, approximately equimolar amounts of N-glycosylated (22 kDa) and unglycosylated (17 kDa) IL-1 beta protein were secreted. The N-glycosylated yeast recombinant IL-1 beta exhibited a 5-7-fold lower specific activity compared to the unglycosylated species. The mechanism responsible for inefficient glycosylation was also studied. We found no differences in secretion kinetics or processing between the two extracellular forms of IL-1 beta. The 17-kDa protein, which was found to lack core sugars, does not result from deglycosylation of the 22-kDa protein in vivo and does not result from saturation of the glycosylation enzymatic machinery through overexpression. Alteration of the uncommon Cys8 residue in the -Asn-X-Ser/Thr-glycosylation site to Ser also had no effect. However, increasing the distance between Asn7 and the signal processing site increased the extent of core N-linked glycosylation, suggesting a reduction in glycosylation efficiency near the NH2 terminus.

Amino Acid Sequence↗
PubMed1991

Structure-function mapping of interleukin 1 precursors. Cleavage leads to a conformational change in the mature protein.

The two interleukin 1 (IL-1) genes (IL-1 alpha and beta) encode 31-kDa precursor molecules, which are cleaved upon secretion to generate the mature, active, carboxyl-terminal 17-kDa proteins. The IL-1 beta precursor is inactive, whereas the IL-1 alpha precursor is as active as the mature IL-1 alpha. In this report, we demonstrate that when either of the recombinant precursors is processed to the mature form, the mature region undergoes a conformational change from a proteinase K-sensitive structure to one that is proteinase K-insensitive. In addition, cysteine residues that are exposed to solvent in the IL-1 beta precursor become buried in the mature protein. Limited structure-activity mapping of the IL-1 beta precursor indicates that the amino-terminal 76 residues are responsible for the conformational change, whereas the most dramatic change in biological activity occurs after further removal of residues 77-94. These findings suggest that the altered structure of the mature region in precursor IL-1s has been conserved for some function. Denaturation/renaturation experiments implicate the precursor domain in protein folding, and by analogy with signal-directed secretory proteins, the unique conformation of the precursors may play a role in IL-1 secretion.

Amino Acid Sequence↗
PubMed1991

Inhibition of the Clostridium perfringens phospholipase C hydrolysis of a thiophosphate analog of lysophosphatidylcholine by micelle-bound ammonium and sulfonium cations.

Cetyltrimethylammonium and n-octadecyldimethylsulfonium bromides inhibit the Clostridium perfringens phospholipase C-catalyzed hydrolysis of 1-S-phosphocholine-2-O-hexadecanoyl-1-mercapto-2-ethanol (1) at pH 7.5, 37 degrees C, mu = 0.15 with KCl. Mixed micelles containing 1 and either inhibitor are substrates for the enzyme and the fraction of activity remaining is a monotonic, but non-linear function of the mole fraction of inhibitor. Simple saturation kinetics are observed as the concentration of 1 is increased in mixed micelles containing a constant mole fraction of inhibitor. Inhibition constants for cetyltrimethylammonium and n-octadecyldimethylsulfonium bromides are 0.66 +/- 0.04 and 0.25 +/- 0.02 mM, respectively. The data suggest that the significant inhibition previously observed for soluble alkyldisulfonium salts (K50 for dodecamethylene-bis(dimethylsulfonium) bromide, 27 microM) is dependent upon bifunctional cationic interactions rather than hydrophobic binding.

Cations↗
PubMed1991

Production of dimer-specific and dengue virus group cross-reactive mouse monoclonal antibodies to the dengue 2 virus non-structural glycoprotein NS1.

A panel of mouse monoclonal antibodies (MAbs) raised against the non-structural glycoprotein NS1 of dengue 2 virus (PR159) was studied for cross-reactivity with the NS1 protein of other dengue virus serotypes and other members of the Flaviviridae using immunoblotting. Most of the 35 anti-NS1 MAbs were found to be specific for dengue 2 virus NS1 (some of which were specific for the native, dimeric form of this protein), but others were found to cross-react within the dengue virus group. This latter group of MAbs, although dominated by MAbs defining a dengue 2 and 4 virus subgroup, also contained some MAbs that were shown to cross-react with both linear (sequential) and conformational epitopes common to the NS1 glycoproteins of all four dengue virus serotypes. Several of these MAbs were also able to cross-react with other flaviviruses, most notably viruses from the Japanese encephalitis antigenic complex. Although cross-reactive epitopes were previously demonstrated on this glycoprotein using polyclonal sera from dengue virus-infected animals and human, this is the first report of the isolation of MAbs which define these determinants and which will allow their further analysis.

Animals↗
PubMed1991

5-lipoxygenase inhibitory activity of zileuton.

Zileuton [N-(1-benzo[b]thien-2-ylethyl)-N-hydroxyure] inhibited 5-hydroxyeicosatetraenoic acid synthesis by rat basophilic leukemia cell 20,000 x g supernatant and rat polymorphonuclear leukocytes (PMNL) (IC50 = 0.5 and 0.3 microM) respectively. It also inhibited leukotriene (LT)B4 biosynthesis by rat PMNL (IC50 = 0.4 microM), human PMNL (IC50 = 0.4 microM) and human whole blood (IC50 = 0.9 microM). Inhibition of human PMNL LTB4 biosynthesis was removed readily by a simple wash procedure. At concentrations up to 100 microM, the compound produced little or no inhibition of several related enzymes, such as platelet 12-lipoxygenase, soybean and rabbit reticulocyte 15-lipoxygenase and sheep seminal vesicle cyclooxygenase. At p.o. doses from 0.5 to 5 mg/kg in the dog, zileuton produced a rapid and sustained inhibition of ex vivo blood LTB4 biosynthesis which correlated with the pharmacokinetic behavior of the compound. In a similar ex vivo study in the rat, the compound displayed an p.o. ED50 of 2 mg/kg. Zileuton was highly effective in preventing 6-sulfidopeptide LT formation in the rat peritoneal cavity triggered by an antigen-antibody reaction with an ED50 of 3 mg/kg. In experimental models of inflammation, zileuton significantly reduced arachidonic-acid induced mouse ear edema (ED50 = 31 mg/kg) and also attenuated inflammatory cell accumulation in the rat pleural Arthus reaction. The effectiveness of this compound for preventing LT formation in vitro, ex vivo and in vivo suggests its utility for preventing the pathophysiological effects of the LTs and other 5-lipoxygenase products in animals and in humans.

Administration, Oral↗
PubMed1990

Secretion of N-glycosylated human recombinant interleukin-1 alpha in Saccharomyces cerevisiae.

We have expressed fragments of the cDNA coding for mature human interleukin-1 alpha (hIL-1 alpha) in Saccharomyces cerevisiae. Mature hIL-1 alpha contains one potential N-linked glycosylation site that is not recognized in mammalian cells. Translational fusions to either one of three yeast signal sequences resulted in secretion of bioactive, N-glycosylated hIL-1 alpha. The extent of glycosylation was significantly reduced using the alpha-factor signal sequence, which itself contains three N-linked glycosylation sites known to be core glycosylated. N-glycosylation has no effect on biological specific activity.

Amino Acid Sequence↗
PubMed1990

Processing of precursor interleukin 1 beta and inflammatory disease.

The processing of precursor interleukin 1 beta (IL1 beta) by elastase, cathepsin G, and collagenase, the major proteases released at sites of inflammation, was investigated using recombinant pro-IL1 beta. Each of these proteases cleaved the 31-kDa inactive precursor to a form similar in size and specific activity (greater than 10(8) units/mg) to the 17-kDa mature protein isolated from activated monocytes. Elastase, collagenase, and cathepsin G cleaved the IL1 beta precursor at distinct sites which are amino-terminal to the monocyte-processing site, Ala-117 (Cameron, P., Lumjuco, G., Rodkey, J., Bennett, C., and Schmidt, J. A. (1985) J. Exp. Med. 162, 790-801). Amino-terminal sequencing of the products of digestion by elastase and cathepsin G determined that resultant active IL1 beta proteins contained an additional 13 or 3 amino acids relative to mature IL1 beta. Synovial fluid collected from patients with inflammatory polyarthritis and bronchoalveolar lavage fluid from patients with sarcoidosis supplied similar processing activity(s). Control fluids from patients who had no symptoms of inflammatory disease did not exhibit processing activity. Lavage fluids that processed precursor IL1 beta were demonstrated to contain cathepsin G and/or elastase activity, whereas controls were negative. Because a significant fraction of IL1 beta may be secreted from monocytes as the inactive 31-kDa precursor (Hazuda, D. J., Lee, J. C., and Young, P. R. (1988) J. Biol. Chem. 263, 8473-8479, Bomford, R., Absull, E., Hughes-Jenkins, C., Simpkin, D., and Schmidt, J. (1987) Immunology 62, 543-549, and Mizel, S. B. (1988) in Cellular and Molecular Aspects of Inflammation Poste, G., and Crooke, S., eds) pp. 75-93, Plenum Publishing Corp., New York), these results suggest that in vivo the IL1 beta precursor can be processed after secretion by any of several proteases released at inflammatory sites.

Amino Acid Sequence↗