Search PubMed⌕ Search

Biomedical subjects

E J Corey

Publications and source records attributed to E J Corey.

At least 109 records · Page 6Linked to original sources
PubMed1994

Molecular cloning, characterization, and overexpression of ERG7, the Saccharomyces cerevisiae gene encoding lanosterol synthase.

We report the cloning, characterization, and overexpression of Saccharomyces cerevisiae ERG7, which encodes lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme responsible for the complex cyclization/rearrangement step in sterol biosynthesis. Oligonucleotide primers were designed corresponding to protein sequences conserved between Candida albicans ERG7 and the related Arabidopsis thaliana cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A PCR product was amplified from yeast genomic DNA using these primers and was used to probe yeast libraries by hybridization. Partial-length clones homologous to the two known epoxysqualene mutases were isolated, but a full-length sequence was found neither in cDNA nor genomic libraries, whether in phage or plasmids. Two overlapping clones were assembled to make a functional reconstruction of the gene, which contains a 2196-bp open reading frame capable of encoding an 83-kDa protein. The reconstruction complemented the erg7 mutation when driven from either its native promoter or the strong ADH1 promoter.

Amino Acid Sequence↗
PubMed1993

Isolation of an Arabidopsis thaliana gene encoding cycloartenol synthase by functional expression in a yeast mutant lacking lanosterol synthase by the use of a chromatographic screen.

Whereas vertebrates and fungi synthesize sterols from epoxysqualene through the intermediate lanosterol, plants cyclize epoxysqualene to cycloartenol as the initial sterol. We report the cloning and characterization of CAS1, an Arabidopsis thaliana gene encoding cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A yeast mutant lacking lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] was transformed with an A. thaliana cDNA yeast expression library, and colonies were assayed for epoxysqualene mutase activity by thin-layer chromatography. One out of approximately 10,000 transformants produced a homogenate that cyclized 2,3-epoxysqualene to the plant sterol cycloartenol. This activity was shown to be plasmid dependent. The plasmid insert contains a 2277-bp open reading frame capable of encoding an 86-kDa protein with significant homology to lanosterol synthase from Candida albicans and squalene-hopene cyclase (EC 5.4.99.-) from Bacillus acidocalcarius. The method used to clone this gene should be generally applicable to genes responsible for secondary metabolite biosynthesis.

Amino Acid Sequence↗
PubMed1991

Hepoxilin A3 (HxA3) is formed by the rat aorta and is metabolized into HxA3-C, a glutathione conjugate.

In this paper we describe the release of hepoxilin A3 (HxA3) by intact pieces of the rat thoracic aorta and its stimulation by exogenous arachidonic acid but not by the calcium ionophore A23187. Homogenates of the rat aorta metabolize HxA3 via two competing pathways; one involves hepoxilin epoxide hydrolase to form the trihydroxy metabolite, trioxilin A3 (TrXA3), and a second pathway involves conjugation of HxA3 with glutathione via glutathione S-transferase to form a glutathione conjugate, which we refer to as hepoxilin A3-C (HxA3-C), a name based upon the accepted nomenclature for the glutathione conjugate leukotriene C. The formation of HxA3-C was dependent on the presence of reduced glutathione in the incubation medium. HxA3-C formation was greatly enhanced in the presence of TCPO, an epoxide hydrolase inhibitor which blocks utilization of the substrate via hepoxilin epoxide hydrolase. Comparison of HxA3-C formation by several arteries and veins indicated that glutathione conjugation was more evident in veins than arteries. The aorta from spontaneously hypertensive rats was essentially similar in HxA3-C formation to aorta from local normotensive Wistar rats although the aorta from the normotensive Wistar Kyoto rats was much more active than aorta from either of the two other rat types. The biological activity of HxA3 and HxA3-C was investigated on isolated helicoidal strips of the rat aorta. While both compounds were inactive on their own, HxA3 and to a lesser extent HxA3-C potentiated the contractile response induced by norepinephrine. The present results provide evidence of the presence in rat aorta of a new pathway of arachidonic acid metabolism whose products may possess potential regulatory properties on vascular tissue.

8,11,14-Eicosatrienoic Acid↗
PubMed1991

Hepoxilin A3 increases vascular permeability in the rat skin.

We have recently shown that hepoxilins are formed by and act on human neutrophils leading to an increase in intracellular levels of calcium and activation of the release of arachidonic acid and diacylglycerol [6, 9]. Since neutrophil activation and accumulation is involved in the inflammatory process resulting in vascular permeability in the rat skin, we investigated the effects of hepoxilins on this process. Hepoxilins administered s.c. resulted in a concentration- and time-dependent leakage of dye to the extravascular compartment of skin from rats to which Evans Blue had been administered. These results were compared to experiments in which prostaglandin E2 was used. The threshold dose of hepoxilin that elicited an effect reached a level of significance over control within 5 min of administration at the 10 ng dose (139% +/- 7%, n = 6). Similar findings were obtained with prostaglandin E2, the level of significance was reached also at 5 min at the 10 ng dose (177% +/- 7% of control, n = 6). The maximum effect observed for both hepoxilin and prostaglandin E2 was 60 min although this did not differ significantly from 30 min except for the highest dose of PGE2 (100 ng). However, the extent of the effect observed for prostaglandin E2 was greater than that for hepoxilin after longer periods, i.e. at 60 min, the prostaglandin E2 effect being 238% +/- 10% of control, and hepoxilin A3 being 167% +/- 10% of control. These results demonstrate that hepoxilins may participate in inflammatory processes.

8,11,14-Eicosatrienoic Acid↗
PubMed1990

Hepoxilin A3 blocks the release of norepinephrine from rat hippocampal slices.

Hepoxilin A3 was previously shown to display neuromodulatory actions on rat hippocampal CA1 neurons, with hyperpolarization of the membrane potential, an increase in the amplitude and duration of the post-spike train after hyperpolarization and an increase in the inhibitory post synaptic potential. The present report describes new biochemical evidence of a presynaptic action of hepoxilin A3 in rat hippocampal slices prelabeled with [3H]-norepinephrine. Hepoxilin A3 on its own had a marginal effect on the release of label, but blocked release which was induced by 4-aminopyridine (4-AP). Prostaglandin E2 also behaved in a similar way. These results demonstrate that hepoxilins modulate neurotransmission in the mammalian CNS through both pre- and postsynaptic actions.

4-Aminopyridine↗
PubMed1990

Isozyme specificity in the conversion of hepoxilin A3 (HxA3) into a glutathionyl hepoxilin (HxA3-C) by the Yb2 subunit of rat liver glutathione S-transferase.

1-14C-Labeled hepoxillin A3 is transformed by a purified preparation of glutathione S-transferase in the presence of glutathione into a glutathionyl conjugate in which the glutathione is covalently coupled to the carbon 11 position of hepoxilin A3. We have termed the glutathione conjugate hepoxilin A3-C in keeping with the established nomenclature for glutathione conjugates in the leukotriene series. Using [3H]glutathione as cosubstrate, the kinetics of the reaction were followed. Among various rat liver glutathione S-transferase isozymes, a homodimer of the Yb2 subunit showed the best activity, while isozymes containing the Ya and Yc subunits showed marginal activity with hepoxilin A3 as substrate.

8,11,14-Eicosatrienoic Acid↗
PubMed1990

Receptor-mediated action of hepoxilin A3 releases diacylglycerol and arachidonic acid from human neutrophils.

We have previously shown that hepoxilin A3 increases the intracellular concentration of Ca+2 in human neutrophils. Herein we address the initial events of hepoxilin action on the neutrophil which precede the rise in intracellular calcium. We show that hepoxilin A3 at 10-1000 nM concentrations releases from [1-14C]-arachidonic acid labeled neutrophils diacylglycerol and unesterified arachidonic acid in a time and concentration dependent fashion. The release of arachidonic acid and diacyglycerol are receptor-mediated events which are blocked by pertussis toxin. This data shows that hepoxilin A3 stimulates phospholipases C and A2 in the cell which may be involved in the rise in cytosolic calcium. Thus, hepoxilins may represent a hitherto unrecognised class of cellular mediators.

8,11,14-Eicosatrienoic Acid↗
PubMed1990

Hepoxilin A3 induces changes in cytosolic calcium, intracellular pH and membrane potential in human neutrophils.

The effects of hepoxilin A3 (HxA3), a 12-lipoxygenase metabolite of arachidonic acid, on cytosolic calcium ([Ca2+]i), intracellular pH (pHi), transmembrane potential and right-angle light scattering in human neutrophils were investigated. A rapid, transient elevation of [Ca2+]i was observed with HxA3 which was dependent on the concentration used. The effect of HxA3 on [Ca2+]i was blocked by pertussis toxin, suggesting involvement of receptors coupled to GTP-binding proteins. Experiments in Ca2(+)-free medium and using intracellular Ca2+ chelators indicated that HxA3 mobilized Ca2+ from intracellular stores. At similar concentrations, HxA3 altered pHi, producing an initial acidification followed by an alkalinization. The initial acidification was decreased in cells loaded with a Ca2+ chelator. In the presence of N-ethyl-N-(1-methylethyl)amino amiloride, an inhibitor of the Na+/H+ antiport, HxA3 induced a greater acidification but failed to elicit the recovery phase, suggesting that the latter is due to activation of the antiport. HxA3 also depolarized the membrane potential, although this effect was small. A decrease in right-angle light scattering, qualitatively similar to that observed with chemotactic peptides, was seen with HxA3, indicating that the 12-lipoxygenase metabolite can induce shape changes in neutrophils. At the concentrations used for the above effects, HxA3 was unable to generate a respiratory burst. These findings suggest that hepoxilins, which are formed by stimulated neutrophils, may have a role as messengers in neutrophil activation.

8,11,14-Eicosatrienoic Acid↗
PubMed1990

Nomenclature.

Explore the source record for details and available documents.

Arachidonic Acids↗
PubMed1990

A glutathione conjugate of hepoxilin A3: formation and action in the rat central nervous system.

Incubation of (8R)- and (8S)-[1-14C]hepoxilin A3 [where hepoxilin A3 is 8-hydroxy-11,12-epoxyeicosa-(5Z,9E,14Z)-trienoic acid] and glutathione with homogenates of rat brain hippocampus resulted in a product that was identified as the (8R) and (8S) diastereomers of 11-glutathionyl hepoxilin A3 by reversed-phase high performance liquid chromatographic comparison with the authentic standard made by total synthesis. Identity was further confirmed by cleavage of the isolated product with gamma-glutamyltranspeptidase to yield the corresponding cysteinylglycinyl conjugate that was identical by reversed-phase high performance liquid chromatographic analysis with the enzymic cleavage product derived from the synthetic glutathionyl conjugate. The glutathionyl and cysteinylglycinyl conjugate are referred to as hepoxilin A3-C and hepoxilin A3-D, respectively, by analogy with the established leukotriene nomenclature. Formation of hepoxilin A3-C was greatly enhanced with a concomitant decrease in formation of the epoxide hydrolase product, trioxilin A3, when the epoxide hydrolase inhibitor trichloropropene oxide was added to the incubation mixture demonstrating the presence of a dual metabolic pathway in this tissue involving hepoxilin epoxide hydrolase and glutathione S-transferase processes. Hepoxilin A3-C was tested using intracellular electrophysiological techniques on hippocampal CA1 neurons and found to be active at concentrations as low as 16 nM in causing membrane hyperpolarization, enhanced amplitude and duration of the post-spike train afterhyperpolarization, a marked increase in the inhibitory postsynaptic potential, and a decrease in the spike threshold. These findings suggest that these products in the hepoxilin pathway of arachidonic acid metabolism formed by the rat brain may function as neuromodulators.

8,11,14-Eicosatrienoic Acid↗
PubMed1989

New products in the hepoxilin pathway: isolation of 11-glutathionyl hepoxilin A3 through reaction of hepoxilin A3 with glutathione S-transferase.

We describe herein the metabolism of hepoxilin A3 (HxA3) by glutathione S-transferase (GST) into a glutathione conjugate. The reaction was carried out with HxA3 (unlabelled and 14C-labelled) and glutathione (unlabelled and tritium labelled). When two isomers of HxA3 were reacted with GST, two products were formed. Only one product was formed when a single isomer of HxA3 was used. The isomeric product HxB3 was marginally active indicating considerable specificity in the reaction with GST. The products were characterized by retention of tritium from glutathione and by comparison of their migration on high performance liquid chromatography with authentic reference compounds. The products bear the structure, 11-glutathionyl HxA3.

8,11,14-Eicosatrienoic Acid↗
PubMed1989

Actions of arachidonic acid and hepoxilin A3 on mammalian hippocampal CA1 neurons.

The effects of arachidonic acid and its lipoxygenase metabolites, the hepoxilins, were investigated in rat hippocampal CA1 neurons in vitro by intracellular electrophysiological recordings. Both arachidonic acid and the hepoxilins cause a hyperpolarization which is sometimes followed by a later depolarization, augment the postspike train long-lasting afterhyperpolarization (AHP) and increase orthodromic inhibitory postsynaptic potentials (IPSPs). These data show that this arachidonic acid metabolic pathway has significant actions on mammalian central neurons, and may represent an important mechanism of neuromodulation.

8,11,14-Eicosatrienoic Acid↗
PubMed1989

Localization of the site of the bronchoconstrictor effects of leukotriene C4 compared with that of histamine in asthmatic subjects.

Although the sulfidopeptide leukotrienes are known to be potent bronchoconstrictors, the relative aerodynamic site of response to these compounds is controversial. We determined the decrease in maximal expiratory flow rates (Vmax) from partial and maximal flow-volume curves in seven asthmatic subjects after inhalation of aerosols of histamine or leukotriene C4 (LTC4) while breathing air or a mixture of 80% helium and 20% oxygen (He/O2). Density dependence (DD) of maximal expiratory flow was determined from partial expiratory flow volume curves by an isovolumic comparison of maximal expiratory flows with subjects breathing He/O2 with those obtained while breathing air. Measurements were made before and after inhalation of aerosols generated from graded concentrations of each constrictor agent. An aerodynamic site of response to LTC4 more central than for histamine was indicated by a significant (p less than 0.02) increase in DD with the former but not with the latter agonist. The ratio of Vmax at 30% vital capacity determined from maximal and partial maneuvers (M/P) was routinely higher at baseline while breathing He/O2 compared to the corresponding values with air, suggesting a degree of peripheral obstruction that was reversed by a deep inhalation. Obstruction induced by LTC4 inhalation resulted in a greater increase in M/P compared with baseline when air was the test gas (p less than 0.02). This was not observed when He/O2 was the test gas. Similar effects on M/P were not induced by histamine aerosol inhalation, consistent with a central airway response to LTC4 that was not affected by volume history.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗
PubMed1988

Potentiation of angiostatic steroids by a synthetic inhibitor of arylsulfatase.

Sodium 2-hydroxy-5-nitro-alpha-toluenesulfonate (HNT, compound II, Fig. 7) is a synthetic inhibitor of arylsulfatase (E.C. 3.1.6.1.). At 1 to 10 mM, HNT increased the clotting time of heparinized rabbit blood by 7-fold. In the 6-day chick embryo, mixtures of heparin and hydrocortisone applied to the chorioallantoic membrane, are known to inhibit specifically the growth of capillary blood vessels. HNT potentiated this antiangiogenic activity in a dose-dependent manner when the concentration of heparin was suboptimal. Potentiation of the antiangiogenic activity of steroids by HNT correlated inversely with the concentration of exogenous heparin. In the absence of exogenous heparin, hydrocortisone did not inhibit angiogenesis. Hydrocortisone and HNT, however, inhibited angiogenesis to the same extent as hydrocortisone and heparin. An arylsulfatase with a Km of 1.5 mM for nitrocatechol sulfate as substrate and a Ki of 10.0 microM for the inhibition of HNT, was identified in the chick embryo chorioallantoic membrane. Preincubation of heparin with a commercially available arylsulfatase caused a 50% reduction in antiangiogenic activity of heparin-steroid mixtures applied to the chorioallantoic membrane. This loss of activity was prevented completely by addition of HNT to the arylsulfatase-heparin incubation mixture. These results suggest that HNT (a) potentiates the anticoagulant function of heparin, (b) prevents the inactivation of antiangiogenic activity of heparin by an endogenous arylsulfatase in the chorioallantoic membrane and by a commercial arylsulfatase, and (c) in the presence of angiostatic steroids can inhibit angiogenesis in the chick embryo without the addition of exogenous heparin. On the basis of these data, we propose that this inhibitor of arylsulfatase acts to potentiate angiostatic steroids by suppressing the desulfation of tissue heparin.

Animals↗
PubMed1987

Water induced dismutation of superoxide anion generates singlet molecular oxygen.

Direct spectroscopic measurement of 1268 nm singlet oxygen emission from KO2 suspensions at room temperature in three non-protonic solvents--CCl4, Cl2FCCClF2, and C6F14 by the action of water is reported. The results clearly show that the singlet oxygen generation is due to a water induced reaction, and suggest that one role of the enzyme superoxide dismutase may be the protection of biological structures, for example, lipid membranes, from degradation by singlet oxygen.

Carbon Tetrachloride↗
PubMed1987

Antiarthritic gold compounds effectively quench electronically excited singlet oxygen.

Although certain gold [Au(I)] compounds have been used effectively in the treatment of rheumatoid arthritis for some years, the molecular basis for such therapeutic action has been unclear. One possible mechanism of the action of Au(I) compounds is that they protect unsaturated membrane lipids and proteins against oxidative degradation caused by activated phagocytes that are not properly regulated. In this study it has been shown that superoxide ion (O-2.), a product of activated phagocytes, can be oxidized to electronically excited singlet oxygen (O1(2)delta g), an agent that is capable of peroxidation of unsaturated fatty acid derivatives. It has also been shown that antiarthritic Au(I) compounds are effective deactivators of O1(2)delta g with quenching constants on the order of 10(7) M-1 sec-1.

Arthritis, Rheumatoid↗