A novel cyclooxygenase metabolite of arachidonic acid

The 8000 X g pellet of rabbit placenta transformed arachidonic acid into a number of lipoxygenase and cyclooxygenase products of known structure. A metabolite was also produced which was inhibited by indomethacin and required calcium for its formation. This compound had a UV absorption maximum at 227 nm under acidic or neutral conditions and gave a bathochromic shift to 281 nm under alkaline conditions. Reduction of this metabolite with sodium borohydride produced prostaglandin (PG) F2 alpha (as determined by mass spectrometry), while catalytic hydrogenation increased the molecular weight by four mass units, indicating the presence of two double bonds. Based on the mass spectrum of the derivatized metabolite, the structure proved to be 9,15-dioxo-11-hydroxyprosta-5,13-dienoic acid. This compound is produced by the term placenta and does not appear to be formed from PGE2, PGF2 alpha, or PGD2. The compound is suppressed by GSH and NADPH, but its formation is not increased by NAD or NADP. PGH2 and PGG2 are not converted to 9,15-dioxo-11-hydroxyprosta-5,13-dienoic acid under similar in vitro incubation conditions. This therefore represents conversion of arachidonate to 9,15-dioxo-11-hydroxyprosta-5,13-dienoic acid through a Ca2+-dependent, non-PG dehydrogenase pathway.     

The enzymatic conversion of arachidonic acid into 8,11,12-trihydroxyeicosatrienoic acid. Resolution of rat lung enzyme into two active fractions

The conversion of arachidonic acid into 8,11,12-trihydroxyeicosatrienoic acid by rat lung high-speed supernatant has been resolved into two separate stages through ammonium sulfate precipitation. The first stage is catalysed by 0-30% ammonium sulfate fraction and converts arachidonic acid and 12-hydroperoxyeicosatetraenoic acid into an intermediate, X. X is subsequently utilized in the second stage by the fraction sedimented at 30-50% saturation in ammonium sulfate to form two isomeric 8,11,12-trihydroxyeicosatrienoic acids.     

The spindle checkpoint: structural insights into dynamic signalling

Chromosome segregation is a complex and astonishingly accurate process whose inner working is beginning to be understood at the molecular level. The spindle checkpoint plays a key role in ensuring the fidelity of this process. It monitors the interactions between chromosomes and microtubules, and delays mitotic progression to allow extra time to correct defects. Here, we review and integrate findings on the dynamics of checkpoint proteins at kinetochores with structural information about signalling complexes.     

Substance P and astrocytes: stimulation of the cyclooxygenase pathway of arachidonic acid metabolism

Substance P (SP) may play an important role in the interactions between the nervous system and the immune system. Astrocytes carry receptors for SP on their surfaces. We examined whether ligand-induced receptor activation would lead to the release of arachidonic acid metabolites. SP (10(-10)-10(-8) M) evokes the formation of prostaglandin E and thromboxane B2 in a dose-dependent manner. Structure-activity studies disclosed that the COOH-terminal peptide sequence of SP is primarily responsible for this biological activity. The generation by astrocytes of arachidonate-derived proinflammatory and immunoregulatory compounds in response to SP receptor activation may be relevant to immunoinflammatory responses within the central nervous system and emphasizes the concept of neuroimmunomodulation.     

Curiosity to kill the KAT (kynurenine aminotransferase): structural insights into brain kynurenic acid synthesis

Kynurenine aminotransferases are pyridoxal-5'-phosphate-dependent enzymes, which catalyze the synthesis of kynurenic acid, a highly neuroactive metabolite whose impairment is associated with a number of severe brain disorders. Crystallographic studies of these enzymes from different organisms, including humans, have revealed distinctive structural traits of type I and type II kynurenine aminotransferases. A striking difference concerns domain swapping of the N-terminal regions, which play equivalent key functional roles in both an unswapped and swapped structure in type I and type II isozymes. Different conformational changes during catalysis create divergent active sites in the two isozymes and affect substrate specificity. Structural investigations indicate intriguing evolutionary relationships and pave the way for the design of isozyme-specific inhibitors, which are of interest for the treatment of catastrophic brain diseases such as Alzheimer's disease and schizophrenia.     

Linoleic acid kinetics and conversion to arachidonic acid in the pregnant and fetal baboon.

Linoleic acid plasma kinetics in pregnant baboons and its conversion to long chain polyunsaturates (LCP) in fetal organs is characterized over a 29-day period using stable isotope tracers. Pregnant baboons consumed an LCP-free diet and received [U-13C]linoleic acid (18:2*) in their third trimester of gestation. In maternal plasma, 18:2* dropped to near baseline by 14 days post-dose, while labeled arachidonic acid (20:4*) plateaued at 10 days at about 70% of total labeled fatty acids. After 2;-5 days, total tracer fatty acids decreased in visceral organs, but increased in the fetal brain. Maximal fetal incorporation of 18:2* was 1;-2 days post-dose; thereafter it dropped while 20:4* increased reciprocally. Labeled 20:4 replaced 18:2* in neural tissues by 5 days post-dose. In liver, kidney, and lung, 20:4* became dominant by 12 days, but in heart the crossover was >29 days. Fetal brain 20:4* plateaued by 21 days at 0. 025% of dose, while fetal liver 20:4* was constant from 1 to 29 days at 0.006% of dose. Under these dietary conditions we estimate that the fetus derives about 50% its 20:4 requirement from conversion of dietary 18:2, with the balance from maternal stores, and conclude that 1) fetal organs accumulate 18:2 within a day of a maternal dose and convert much of it to 20:4 within weeks, 2) modest dietary 18:2 levels may support fetal brain requirements for 20:4, and 3) the brain retains n;-6 fatty acids uniquely compared with major visceral organs.     

Molecular dynamics simulations of arachidonic acid complexes with COX-1 and COX-2: insights into equilibrium behavior

The cyclooxygenase (COX) enzymes are responsible for the committed step in prostaglandin biosynthesis, the generation of prostaglandin H(2). As a result, these enzymes are pharmacologically important targets for nonsteroidal antiinflammatory drugs, such as aspirin and newer COX-2 selective inhibitors. The cyclooxygenases are functional homodimers, and each subunit contains both a cyclooxygenase and a peroxidase active site. These enzymes are quite interesting mechanistically, as the conversion of arachidonic acid to prostaglandin H(2) requires two oxygenation and two cyclization reactions, resulting in the formation of five new chiral centers with nearly absolute regio- and stereochemical fidelity. We have used molecular dynamics (MD) simulations to investigate the equilibrium behavior of both COX-1 and COX-2 enzyme isoforms with bound arachidonate. These simulations were compared with reference simulations of arachidonate in solution to explore the effect of enzyme on substrate conformation and positioning in the active site. The simulations suggest that the substrate has greater conformational freedom in the COX-2 active site, consistent with the larger COX-2 active site volume observed in X-ray crystal structures. The simulations reveal different conformational behavior for arachidonate in each subunit over the course of extended equilibrium MD simulations. The simulations also provide detailed information for several protein channels that might be important for oxygen and water transport to or from active sites or for intermediate trafficking between the cyclooxygenase and peroxidase active sites. The detailed comparisons for COX-1 versus COX-2 active site structural fluctuations may also provide useful information for design of new isozyme-selective inhibitors.     

Differential assembly of human tau isoforms in the presence of arachidonic acid

Six tau isoforms arise from the alternative splicing of a single gene in humans. Insoluble, filamentous deposits of tau protein occur in a number of neurodegenerative diseases, and in some of these diseases, the deposition of polymers enriched in certain tau isoforms has been documented. Because of these findings, we have undertaken studies on the efficacy of fatty acid-induced polymerization of the individual tau isoforms found in the adult human CNS. The polymerization of each tau isoform in the presence of two concentrations of arachidonic acid indicated that isoforms lacking N-terminal exons e2 and e3 formed small, globular oligomers that did not go on to elongate into straight (SF) or paired helical (PHF) filaments under our buffer conditions. The polymerization of all isoforms containing e2 or e2 and e3 occurred readily at a high arachidonic acid concentration. Conversely, at a lower arachidonic acid concentration, only tau isoforms containing four microtubule binding repeats assembled well. Under all buffer conditions employed, filaments formed from three of the isoforms containing e2 and e3 resembled SFs in morphology but began to form PHF-like structures following extended incubation at 37 degrees C. These results indicate that polymerization of the intact tau molecule may be facilitated by e2 and e3. Moreover, tau isoforms containing three versus four microtubule binding repeats display different assembly properties depending on the solvent conditions employed.     

Conversion of arachidonic acid to cyclooxygenase and lypoxygenase products,and incorporation into phospholipids in the mouse neuroblastoma clone,Neuro-2A

Arachidonic acid (AA) incorporation into phospholipids and cyclooxygenase and lipoxygenase mediated metabolism of arachidonic acid were studied in homogenized and intact Neuro-2A cells. When 3H8-AA was added to homogenized cells and incubated 20 minutes, 39% of the label was converted to prostaglandins (PGs), 10% to hydroxy-eicosatetraenoic acid (HETE) and 26% was incorporated into phospholipids. PGE2 and PGF2a were the major PGs produced. Synthesis of PGs was blocked by 10 microM indomethacin and synthesis of PGs and HETE was blocked by 10 microM eicosatetraynoic acid (ETYA). The cell homogenate produced the 13,14-dihydro-15-keto metabolites of PGE2 and PGF2a from 3H8-AA and also converted exogenous 3H7-PGE2 and 3H8-PGF2a to metabolites. When intact cells were labeled for 24 hours with 14C1-AA and the cells and media then analyzed, 75% of the radioactivity was incorporated into cellular phospholipids, 0.8% was converted to PGs and metabolites and 0.7% converted to HETE. Cells prelabeled for 24 hours were washed and incubated for 30 minutes in fatty acid free media. There was a 23% release of AA from phospholipids. One-fifth of the released AA was converted to HETE. PG synthesis in the intact resting cells was low. In summary, the Neuro-2A cell provides a good model system for studying arachidonic acid metabolism and incorporation into phospholipids in cells of neuronal origin.     

Effect of various prostaglandins on the release of arachidonic acid from cultured fibroblasts

Effect of various prostaglandins on the release of arachidonic acid from [¹⁴C]arachidonic acid labeled fibroblasts was studied. Prostaglandin(PG) F_2α was found to enhance the release of radioactive arachidonic acid from the cells. The stimulatory effect was dose dependent, and was greater than that of bradykinin. The active compounds can be ranked in potency for the release of arachidonic acid from the pre-labeled cells per cent of control: PGF_2α(200.1%)>PGF_1α (141.8%)>PGD₂ (137.1%)>thromboxane B₂ (113.7%)>PGE₂ (109.4%). On the other hand, PGI₂ showed a strong inhibitory effect on the arachidonic acid release from the pre-labeled cells (the value was only 69% of the control), while 6-ketoPGF_1α, an end metabolite of PGI₂, had no effect.     

Effects of arachidonic acid and prostaglandins on lung function in the intact dog

The effects of the prostaglandin (PG) precursor, arachidonic acid (AA), and the primary PG's, PGF2alpha, and PGD2, on lung function were compared in 39 intact-chest, paralyzed, artificially ventilated dogs. Intravenous AA decreased dynamic compliance (Cdyn) and functional residual capacity and increased airway resistance (Rl) and transpulmonary pressure at end-passive expiration. The decrease in Cdyn correlated closely with a rise in pulmonary arterial pressure (Ppa). Indomethacin abolished airway and vascular responses to AA, but did not attenuate responses to the PG's. The effects of AA, PGD2, and PGF2alpha on lung function and Ppa were similar, whereas PGE2 had little effect. Vagotomy attentuated the RL increase in response to AA, PGE2alpha, and PGD2 and the Cdyn decrease in response to the PG's. The effects of the PG's on compliance were greater than those produced by mechanically increasing pulmonary venous pressure. The present studies suggest that the PG precursor is rapidly converted to agents that have marked effect on both pulmonary vessels and airways, particularly peripheral airways, in the dog.     

Cleave to leave: structural insights into the dynamic organization of the nuclear pore complex

A detailed understanding of the fine structure of the nuclear pore complex has remained elusive. Now, studies on a small protein domain have shed light on the dynamic organization of this massive assembly.     

Outer membrane translocons: structural insights into channel formation

Gram-negative bacteria need to maintain the integrity of their outer membrane while also regulating the secretion of toxins and other macromolecules. A variety of dedicated outer membrane proteins (OMPs) facilitate this process. Recent structural work has shown that some of these proteins adopt classical β-barrel transmembrane structures and rely on structural changes within the barrel lumen to allow passage of substrate proteins. Other secretion systems have OMP components which use transmembrane α-helices and appear to function in a different way. Here we review a selection of recent structural studies which have major ramifications for our understanding of the passage of macromolecules across the outer membrane.     

Bioconversion of arachidonic acid to prostaglandins and related compounds in human myometrium and uterine cervix

Arachidonic Acid metabolites in human myometrium and uterine cervix were studied using silicic acid column chromatography, thin layer chromatography, reversed phase partition chromatography and gas-liquid chromatography. Myometrium produced 6-ketoPGF1 alpha, PGF2 alpha, PGE2, thromboxane B2. Uterine cervix produced 6-ketoPGF1 alpha, PGF2 alpha, PGE2, thromboxane B2, and one hydroxyacid. There was no difference between the rate of conversion of prostaglandins in myometrium and cervix. But only cervix could convert arachidonic acid to hydroxyacid.     

Engineering of a protein with cyclooxygenase and prostacyclin synthase activities that converts arachidonic acid to prostacyclin

Prostacyclin (PGI2), a vascular protector with vasodilation and antithrombotic properties, is synthesized by coupling reactions of cyclooxygenase (COX, the first enzyme) with PGI2 synthase (PGIS, the second enzyme) using arachidonic acid (AA) as an initial substrate. The first COX product, prostaglandin H2 (PGH2) is also a command substrate for other prostanoid enzymes that produce distinct eicosanoids, such as thromboxane A2 (TXA2). The actions of TXA2 to cause vasoconstriction and platelet aggregation oppose the vasodilatory and anti-aggregatory effects of PGI2. Specifically upregulating PGI2 biosynthesis is an ideal model for the prevention and treatment of the TXA2-mediated thrombosis involved in strokes and myocardial infarctions. Here, we report that a single protein was constructed by linking COX-2 and PGIS together to form a new fusion enzyme through a transmembrane domain with 10 or 22 residues. The engineered protein expressed in HEK293 and COS-7 cells was able to continually convert AA to prostaglandin (PG) G2 (catalytic step 1), PGH2 (catalytic step 2), and PGI2 (catalytic step 3). The studies first demonstrate that a single protein with three catalytic functions could directly synthesize PGI2 from AA with a Km of approximately 3.2 microM. Specific upregulation of PGI2 biosynthesis through expression of the engineered single protein in the cells has shown strong activity in inhibiting platelet aggregation induced by AA in vitro, which creates a great potential for the fusion enzyme to be used as one of the new therapeutic interventions for strokes and heart attacks. The studies have also provided a model linking COX with its downstream enzymes to specifically regulate biosynthesis of eicosanoids which have potent biological functions.     

Identification of an unusual cyclooxygenase metabolite of arachidonic acid in rabbit renal medulla

A renal medulla 100,000g pellet metabolized arachidonic acid, C_20:4, to the previously described prostaglandins prostaglandin E₂, 6-ketoprostaglandin F_1α, thromboxane B₂, 12-hydroxyheptadecatrienoic acid, and 11-hydroxyeicosatetraenoic acid. In addition, under conditions of low enzyme to substrate ratios, the renal medulla also produced an unusual metabolite from arachidonic acid. This metabolite was inhibited by indomethacin, and thus suggested that it was a product of the cyclooxygenase. Addition of GSH to the incubation inhibited its formation, while p-hydroxymercuri-benzoate enhanced its formation. This compound was identified by HPLC purification, uv absorption, and gas chromatography-mass spectroscopy. The compound was 9,15 dioxo,11-hydroxyprosta-5,13-dienoic acid.     

Liquid chromatographic-mass spectrometric determination of cyclooxygenase metabolites of arachidonic acid in cultured cells

A liquid chromatographic-electrospray ionization-mass spectrometric (LC-ESI-MS) technique was developed to simultaneously determine the cyclooxygenase metabolites of arachidonic acid (6-keto-PGF(1alpha), PGD(2), PGE(2), PGF(2alpha), and PGJ(2)) produced by cultured cells. Samples were separated on a C(18) column with water-acetonitrile mobile phase, ionized by electrospray, and detected in the positive mode. Selected ion monitoring (SIM) of m/z 353, 335, 335, 319, and 317 were used for quantifying 6-keto-PGF(1alpha), PGD(2), PGE(2), PGF(2alpha), and PGJ(2), respectively. Prostaglandins were detected at concentrations as low as 1 pg (S/N=3) on the column. The method was used to determine the production of PGs from bovine coronary artery endothelial cells (ECs) and human prostate cancer cells (PC-3) with different degree of invasiveness. Bradykinin (10(-6) M) stimulated a marked increase in the production of 6-keto-PGF(1alpha), PGE(2), and PGF(2alpha) and a small increase of PGD(2) by ECs. 6-Keto-PGF(1alpha) was the major metabolite in these cells. The production of PGE(2) was threefold higher and PGD(2) was twofold higher in PC-3-S (invasive) cells than in PC-3-U (non-invasive) cells.     

Dissecting the fibrillin microfibril: structural insights into organization and function

Force-bearing tissues such as blood vessels, lungs, and ligaments depend on the properties of elasticity and flexibility. The 10 to 12 nm diameter fibrillin microfibrils play vital roles in maintaining the structural integrity of these highly dynamic tissues and in regulating extracellular growth factors. In humans, defective microfibril function results in several diseases affecting the skin, cardiovascular, skeletal, and ocular systems. Despite the discovery of fibrillin-1 having occurred more than two decades ago, the structure and organization of fibrillin monomers within the microfibrils are still controversial. Recent structural data have revealed strategies by which fibrillin is able to maintain its architecture in dynamic tissues without compromising its ability to?interact with itself and other cell matrix components. This review summarizes our current knowledge of microfibril structure, from individual fibrillin domains and the calcium-dependent tuning of pairwise interdomain interactions to microfibril dynamics, and how this relates to microfibril function in health and disease.     

Effects of prostaglandins and arachidonic acid on baboon cerebral and mesenteric arteries

Effects of prostaglandins (PGs) E1, E2, F2 alpha and I2 in a wide range of concentration were examined in mesenteric and cerebral arteries isolated from mature baboons. PGs E1, E2 and F2 alpha at low concentrations (10(-10) to 10(-7) M) elicited relaxation in helically cut strips of cerebral arteries precontracted with phenylephrine. In contrast, the PGs did not cause relaxation in the mesenteric artery. PGI2 (10(-9) to 10(-6) M) produced marked relaxation in both arteries. The EC25 for PGI2 in the mesenteric artery was significantly lower than that in the cerebral artery. During baseline conditions, cerebral arteries contracted in response to high concentrations (greater than 10(-7) M) of PGs E1, E2 and F2 alpha. In mesenteric arteries, a large contraction was induced by PGs F2 alpha and E2 but not by PGE1. Arachidonic acid (10(-6) M) produced an aspirin-inhibitable relaxation in both arteries to a similar extent, so that the vasodilator PG(s) formed in the two different arterial walls appear to exert a similar relaxant action. Thus, the baboon mesenteric artery was more sensitive to PGI2 for the relaxant effect than was the cerebral artery, while PGs F2 alpha, E1 and E2 caused only a contraction in the mesenteric artery but both relaxation and contraction in the cerebral artery.