Isomer-selected ion-molecule reactions of acetylene cations with propyne and allene.

One of the fundamental goals of chemistry is to determine how molecular structure influences interactions and leads to different reaction products. Studies of isomer-selected and resolved chemical reactions can shed light directly on how form leads to function. In the following, we present the results of gas-phase reactions between acetylene cations (C2D2+) with two different isomers of C3H4: propyne (DC3D3) and allene (H2C3H2). Our highly controlled, trapped-ion environment allows for precise determination of reaction products and kinetics. From these results, we can infer details of the underlying reaction dynamics of C2H2+ + C3H4. Through the synergy of experimental results and high-level quantum chemical potential energy surface calculations, we are able to identify distinct reaction mechanisms for the two isomers. We find long-range charge exchange with no complex formation is favored for allene, whereas charge exchange leads to an intermediate reaction complex for propyne and thus, different products. Therefore, this reaction displays a pronounced isomer-selective bi-molecular reactive process.

A multi-level approach of evaluating crew resource management training: a laboratory-based study examining communication skills as a function of team congruence.

The article proposes a multi-level approach for evaluating communication skills training (CST) as an important element of crew resource management (CRM) training. Within this methodological framework, the present work examined the effectiveness of CST in matching or mismatching team compositions with regard to hierarchical status and competence. There is little experimental research that evaluated the effectiveness of CRM training at multiple levels (i.e. reaction, learning, behaviour) and in teams composed of members of different status and competence. An experiment with a two (CST: with vs. without) by two (competence/hierarchical status: congruent vs. incongruent) design was carried out. A total of 64 participants were trained for 2.5 h on a simulated process control environment, with the experimental group being given 45 min of training on receptiveness and influencing skills. Prior to the 1-h experimental session, participants were assigned to two-person teams. The results showed overall support for the use of such a multi-level approach of training evaluation. Stronger positive effects of CST were found for subjective measures than for objective performance measures. STATEMENT OF RELEVANCE: This work provides some guidance for the use of a multi-level evaluation of CRM training. It also emphasises the need to collect objective performance data for training evaluation in addition to subjective measures with a view to gain a more accurate picture of the benefits of such training approaches.

Protein serine/threonine phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 to modulate its transcriptional and apoptotic activities.

We have previously demonstrated that the serine/threonine protein phosphatase-1 (PP-1) plays an important role in promoting cell survival. However, the molecular mechanisms by which PP-1 promotes survival remain largely unknown. In the present study, we provide evidence to show that PP-1 can directly dephosphorylate a master regulator of apoptosis, p53, to negatively modulate its transcriptional and apoptotic activities, and thus to promote cell survival. As a transcriptional factor, the function of p53 can be greatly regulated by phosphorylation and dephosphorylation. While the kinases responsible for phosphorylation of the 17 serine/threonine sites have been identified, the dephosphorylation of these sites remains largely unknown. In the present study, we demonstrate that PP-1 can dephosphorylate p53 at Ser-15 and Ser-37 through co-immunoprecipitation, in vitro and in vivo dephosphorylation assays, overexpression and silence of the gene encoding the catalytic subunit for PP-1. We further show that mutations mimicking constitutive dephosphorylation or phosphorylation of p53 at these sites attenuate or enhance its transcriptional activity, respectively. As a result of the changed p53 activity, expression of the downstream apoptosis-related genes such as bcl-2 and bax is accordingly altered and the apoptotic events are either largely abrogated or enhanced. Thus, our results demonstrate that PP-1 directly dephosphorylates p53, and dephosphorylation of p53 has as important impact on its functions as phosphorylation does. In addition, our results reveal that one of the molecular mechanisms by which PP-1 promotes cell survival is to dephosphorylate p53, and thus negatively regulate p53-dependent death pathway.

Production and physiological actions of anandamide in the vasculature of the rat kidney.

The endogenous cannabinoid receptor agonist anandamide is present in central and peripheral tissues. As the kidney contains both the amidase that degrades anandamide and transcripts for anandamide receptors, we characterized the molecular components of the anandamide signaling system and the vascular effects of exogenous anandamide in the kidney. We show that anandamide is present in kidney homogenates, cultured renal endothelial cells (EC), and mesangial cells; these cells also contain anandamide amidase. Reverse-transcriptase PCR shows that EC contain transcripts for cannabinoid type 1 (CB1) receptors, while mesangial cells have mRNA for both CB1 and CB2 receptors. EC exhibit specific, high-affinity binding of anandamide (Kd = 27.4 nM). Anandamide (1 microM) vasodilates juxtamedullary afferent arterioles perfused in vitro; the vasodilation can be blocked by nitric oxide (NO) synthase inhibition with L-NAME (0.1 mM) or CB1 receptor antagonism with SR 141716A (1 microM), but not by indomethacin (10 microM). Anandamide (10 nM) stimulates CB1-receptor-mediated NO release from perfused renal arterial segments; a similar effect was seen in EC. Finally, anandamide (1 microM) produces a NO-mediated inhibition of KCl-stimulated [3H]norepinephrine release from sympathetic nerves on isolated renal arterial segments. Hence, an anandamide signaling system is present in the kidney, where it exerts significant vasorelaxant and neuromodulatory effects.

An ion trap time-of-flight mass spectrometer with high mass resolution for cold trapped ion experiments.

Trapping molecular ions that have been sympathetically cooled with laser-cooled atomic ions is a useful platform for exploring cold ion chemistry. We designed and characterized a new experimental apparatus for probing chemical reaction dynamics between molecular cations and neutral radicals at temperatures below 1 K. The ions are trapped in a linear quadrupole radio-frequency trap and sympathetically cooled by co-trapped, laser-cooled, atomic ions. The ion trap is coupled to a time-of-flight mass spectrometer to readily identify product ion species and to accurately determine trapped ion numbers. We discuss, and present in detail, the design of this ion trap time-of-flight mass spectrometer and the electronics required for driving the trap and mass spectrometer. Furthermore, we measure the performance of this system, which yields mass resolutions of m/Δm ≥ 1100 over a wide mass range, and discuss its relevance for future measurements in chemical reaction kinetics and dynamics.

N-Acylation of ethanolamine phospholipids by acyl transfer does not involve hydrolysis.

N-Acylethanolamine phospholipids occur in infarcted but not in normal canine myocardium. Their synthesis is catalyzed by a membrane-bound, Ca2+-requiring N-acyltransferase (transacylase) which transfers acyl groups from the sn-1 position of various phospholipids including phosphatidylethanolamine to the amino group of ethanolamine phospholipids. When dog heart mitochondria are incubated in media containing Ca2+ and H2(18)O, the resulting N-acylethanolamine phospholipids do not accumulate 18O in either the amide or 1-O-acyl groups. The results indicate that acyl transfer occurs without hydrolysis, most likely through an acyl-enzyme complex which may be covalently linked.

N-acylethanolamine phospholipid metabolism in normal and ischemic rat brain.

N-Acylethanolamine phospholipids accumulate in rat brain during post-decapitative ischemia. Small amounts of these phospholipids consisting primarily of diacyl and alkenylacyl species can be detected within 15 min of ischemia and they increase linearly for 60 min. This ischemia-induced synthesis is more pronounced in developing rat brain (approx. 5.0 nmol/h per mumol lipid P) than in adult brain (0.4 nmol). Pulse labeling experiments with subcellular preparations of 10-day-old rat brain indicate a precursor-product relationship between ethanolamine phospholipids and their N-acyl analogs. N-Acylation of endogenous substrates occurs with both microsomes and mitochondria, exhibits a pH optimum of 10 and requires 1 mM Ca2+ for maximal (0.2 mM Ca2+ for half maximal) activity. Cell-free preparations of both developing and adult rat brain contain a phosphodiesterase which hydrolyzes N-acylphosphatidylethanolamine to phosphatidic acid and N-acylethanolamine. The latter is further hydrolyzed to fatty acid and ethanolamine by an amidohydrolase. [1-3H]Ethanolamine, injected intracerebrally or intraperitoneally into 13- and 18-day-old rats, is incorporated into brain ethanolamine phospholipids. Since small amounts of radioactivity are also associated with N-acylethanolamine phospholipids 5 and 24 h after injection of the substrate, it appears that these phospholipids may occur at a very low level as a natural lipid constituent of rat brain.

Alterations of fatty acyl turnover in macrophage glycerolipids induced by stimulation. Evidence for enhanced recycling of arachidonic acid.

Glycerophospholipid biosynthesis by the de novo pathway was assessed in mouse peritoneal macrophages by pulse-labeling with [U-14C]glycerol. Phosphatidylcholine (PC), which amounts to about 35% of total cellular phospholipids, exhibited the highest rate of glycerol uptake, followed by phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Remodeling of PC molecular species by deacylation/reacylation was established by determining the redistribution of glycerol label over 2 h after a 1 h pulse of [U-14C]glycerol and by determining incorporation of 18O from H2(18)O-containing media. These data suggest that stearic and arachidonic acid enter PC primarily by the remodeling pathway but that small amounts of highly unsaturated molecular species, including 1,2-diarachidonoyl PC, are rapidly synthesized de novo, and subsequently remodeled or degraded. Treatment of the cells with the ionophore A23187 resulted in the selective enhancement of arachidonate turnover in PC, PI and neutral lipid, as well as enhanced de novo PI synthesis. [U-14C]Glycerol labeling experiments suggest that arachidonic acid liberated by Ca(2+)-dependent phospholipase A2 activity is also reacylated in part through de novo glycerolipid biosynthesis, leading to the formation and remodeling of 1,2-diarachidonoyl PC and other highly polyunsaturated molecular species.

The role of cardiolipin as an acyl donor in dog heart N-acylethanolamine phospholipid biosynthesis.

N-Acylethanolamine phospholipids were produced from endogenous substrates with dog heart mitochondrial and microsomal preparations. With mitochondria the N-acyl group contained 13.8% linoleate, with microsomes only 3.6%. Cardiolipin comprised 18.5% of mitochondrial and 3.3% of microsomal lipid P and contained 93.7 and 72.4% linoleic acid, respectively. Incubation of dog heart subcellular fractions with [1-14C]linoleoyl cardiolipin in the presence of Ca2+ resulted in the formation of N-acylethanolamine phospholipids labeled primarily in the N-acyl and 1-O-acyl moieties. The data indicate that cardiolipin is the major source of linoleic acid used in the N-acylation of ethanolamine phospholipids by transacylase activity.

N-Acylation of dog heart ethanolamine phospholipids by transacylase activity.

Radioactive N-acylethanolamine phospholipids were produced in dog heart homogenates incubated with acyl-labeled phosphatidylcholine in the presence of 5 mM Ca2+ and Triton X-100. 70-80% of the label in the N-acylethanolamine phospholipids was recovered in the N-acyl groups and most of the remainder was in the 1-O-acyl groups. Incubations with 1,2-dipalmitoylPC and 1-palmitoyl-2-linoleoylPC labeled in either the 1-O-acyl or 2-O-acyl moiety showed the predominant utilization of the acyl groups at the sn-1 position, indicating transacylation by phospholipase A1 (or lysophospholipase) activity. It is suggested that intramolecular transacylation from 1-O-acyl to N-acyl groups of phosphatidylethanolamine also occurred to some extent, thus providing a free primary hydroxy group as an additional acyl acceptor for the transacylation reaction.

Accumulation of N-acylethanolamine glycerophospholipids in infarcted myocardium.

A new phospholipid was detected in the infarcted areas of canine myocardium 24 h after ligation of the left descending branch of the coronary artery. Both the central and peripheral areas of the infarct contained significant amounts of this lipid (4-6& of lipid phosphorus), but it was not present in the apparently normal portions of the same tissue. The lipid was isolated and characterized by its infrared spectrum, by chemical degradation and by comparison with synthetic and semi-synthetic standards. It was identified as a mixture of N-acylethanolamine glycerophospholipids consisting of diacyl-, alk-1-enylacyl- and alkylacylglycerol species. The N-acyl groups were mainly 16 : 0 and 18 : 0 with smaller proportions of 18 : 1 and 18 : 2. The relative decrease of ethanolamine glycerophospholipids in the infarcated areas and the similarity in their molecular species suggests a percursor-product relationship between these phospholipids.

N-Acylation of ethanolamine phospholipids in canine myocardium.

N-Acylethanolamine phospholipids, which are found in infarcted but not in normal canine myocardium, were produced by preparations of normal myocardial tissue incubated in the presence of millimolar concentrations of Ca2+. Biosynthetic activity from endogenous substrates was associated with both microsomal and mitochondrial fractions and exhibited an alkaline pH optimum. The time course of N-acylethanolamine phospholipid synthesis and degradation was followed after pulse labeling of myocardial ethanolamine phospholipids with [1,2-14C]ethanolamine. Enzymic N-acylation of both phosphatidylethanolamine and lysophosphatidylethanolamine was demonstrated by incubating these substrates with homogenates of myocardial tissue. Neither free fatty acids nor acyl-CoA derivatives served as acyl donor but some of the constituent fatty acids of exogenous phosphatidylethanolamine were recovered among the amide-linked fatty acids of the N-acylethanolamine phospholipids. N-Acylation may thus occur by the transfer of O-acyl groups catalyzed by a Ca2+ -dependent transacylase. Since N-acylethanolamine phospholipids are precursors of the biologically active N-acylethanolamines, their biosynthesis may constitute an important injury-induced metabolic event aimed at the protection of ischemic myocardial tissue.

Structure and metabolism of phospholipids in bovine epididymal spermatozoa.

Lipid analysis of bovine epididymal spermatozoa showed relatively large amounts of alkylacyl- and alk-1-enylacylglycerols in their choline and ethanol-amine phospholipids and alkylacylglycerol as the major constituent of a glycolipid tentatively identified as a monogalactosyl sulfate. The ether lipids exhibited remarkably simple molecular structures, i.e., the phospholipids had only 16:0 as alkyl and alk-1-enyl groups and their constituent fatty acids were almost exclusively 22:5 (n-6) and 22:6 (n-3). The glycolipid had mainly 16:0 as both alkyl and acyl moieties. In contrast, the diacyl choline and ethanolamine phosphoglycerides exhibited a much more complex fatty acid composition. 1,2-Diacylglycerols were the major nonpolar glycerolipid class and their acyl groups consisted almost exclusively of 14:0, 16:0 and 18:0. Labeled glycerol and dihydroxyacetone added to the incubation medium were readily incorporated into sperm lipids under both aerobic and anaerobic conditions. In each case, diacylcholine phosphoglycerides, diacylglycerols and phosphatidic acid were the major labeled lipids. Distribution of label among the molecular species of diacylglycerols and choline phosphoglycerides resembled somewhat their natural abundance. No radioactivity was found in alkylacyl or alk-1-enylacyl glycerolipids. The ether lipids may provide stable structural components of sperm membrane while the diacyl analogs undergo degradation and resynthesis.

On the biosynthesis and metabolism of N-acylethanolamine phospholipids in infarcted dog heart.

Minced tissues from both infarcted and apparently normal areas of canine myocardium 6 h after ligation of the left descending branch of the coronary artery were incubated with [1,2-(14)C]ethanolamine. In each case substantial amounts of radioactivity were incorporated into both phosphatidylethanolamine and N-acylphosphatidylethanolamine. Incubation of homogenates from the same tissues with N-[1-(14)C]palmitoyl phosphatidylethanolamine yielded labeled N-palmitoyl ethanolamine. The data support the concept that N-acylethanolamines, pharmacologically active compounds which accumulate in infarcted myocardium, are produced by N-acylation of ethanolamine phospholipids followed by phosphodiesterase action.

Agonist-stimulated glycerophospholipid acyl turnover in alveolar macrophages.

The inflammatory compounds beta-glucan, a particulate agonist, and tannin, a soluble agonist, are present in cotton dust and, when inhaled, cause massive arachidonic acid release from alveolar macrophages. Earlier work had shown that these agonists exhibit different effects on arachidonate liberation and release, and that only tannin inhibits the uptake and incorporation of exogenous arachidonic acid, suggesting inhibition of reacylation. Here we have used the time-dependent incorporation of 18O from H218O-containing media into glycerophospholipid acyl groups as an indicator of acyl turnover in resting and agonist-treated rabbit alveolar macrophages. Highest turnover rates were seen in phosphatidylinositol ( approximately 30% per hour) and in choline phospholipids (10-20% per hour). Both beta-glucan and tannin stimulated acyl turnover, especially arachidonic acid turnover, in these and other lipid classes by a factor of 2 or more. We conclude that neither agonist promotes arachidonic acid accumulation in and release from alveolar macrophages by inhibiting reacylation.

Properties of canine myocardial phosphatidylethanolamine N-acyltransferase.

Dog heart contains a membrane bound N-acyltransferase (transacylase) which transfers acyl groups from the sn-1 position of membrane phospholipids to the amino group of ethanolamine phospholipids in the presence of millimolar Ca2+ concentrations. Using crude membrane preparations, we found this N-acyltransferase activity to be heat sensitive and inhibited by sulfhydryl reagents. Pretreatment of a membrane fraction with trypsin reduced N-acyltransferase activity to 60% while pretreatment with trypsin and Triton X-100 together reduced it to 30% of the control value. At pH 8.0 both Sr2+ and Mn2+ could fully substitute for Ca2+ with respect to optimum ion concentration and molecular species of the product formed in dog heart membranes from endogenous substrates. Ba2+ was equally effective in achieving N-acylation of ethanolamine phospholipids while other divalent cations were less effective or ineffective. The reaction exhibited a pH optimum of 8.5 to 9.0 with both Ca2+ and Sr2+ while Mn2+ precipitated above pH 8.0 resulting in decreased N-acylation activity. Both phosphatidylcholine and 1-acyl lysophosphatidylcholine could serve as acyl donors. Triton X-100 at a concentration of 0.1% stimulated acyl transfer from exogenous phosphatidylcholine but inhibited acyl transfer from lysophosphatidylcholine.

Occurrence of N-acylethanolamine phospholipids in fish brain and spinal cord.

N-Acylethanolamine phospholipids were identified in the central nervous system of the fresh water fish, pike (Esox lucius) and carp (Cyprinus carpio), at levels ranging from 0.1 to 0.9% of total phospholipid. The N-acylethanolamine phospholipids of carp brain were isolated and characterized by chemical, biochemical and spectroscopic methods. Two major species, 1,2-diacyl-sn-glycero-3-phospho(N-acyl)ethanolamines (approx. 30%) and 1-O-(1'-alkenyl)-2-acyl-sn-glycero-3-phospho(N-acyl)ethanolamines (approx. 70%) were identified. The N-acyl groups of each species consisted primarily of 16:0 (approx. 60%) but also contained 16:1, 18:0 and 18:1 (approx. 10% each) and a number of trace constituents. The N-acylethanolamine phospholipids had O-acyl and O-alkenyl group compositions similar but not identical to those of the ethanolamine phospholipids of the same tissue. N-Acylethanolamine phospholipids were present in all subcellular fractions of carp brain, except mitochondria.

Activation of PAF receptors results in enhanced synthesis of 2-arachidonoylglycerol (2-AG) in immune cells.

The endocannabinoid signaling system is believed to play a down-regulatory role in the control of cell functions. However, little is known about the factors activating endocannabinoid synthesis and which of two known endocannabinoids, 2-arachidonoylglycerol (2-AG) or N-arachidonoylethanolamine (20:4n-6 NAE, anandamide), is of physiological importance. We approached these questions by studying a possible link between cell activation with 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor, PAF) and the generation of 2-AG and anandamide in human platelets and mouse P388D1 macrophages. Human platelets responded to stimulation with the production of various 1- and 2-monoacylglycerols, including 2-AG, whereas stimulation of P388D1 macrophages induced the rapid and selective generation of 2-AG, which was immediately released into the medium. The effect of PAF was receptor mediated, as PAF receptor antagonist BN52021 blocked the effect. The treatment did not change the content of anandamide in either macrophages or platelet-rich plasma. The inhibitors of PI- and PC-specific phospholipases C (U73122 and D609) as well as PI3-kinase inhibitor (wortmannin) attenuated PAF-induced 2-AG production in macrophages. These data suggest a direct role for the endocannabinoid system in controlling immune cell activation status and indicate that 2-AG rather than anandamide is the endocannabinoid rapidly produced in response to proinflammatory stimulation of immune cells.

Biosynthesis and turnover of anandamide and other N-acylethanolamines in peritoneal macrophages.

Polyunsaturated N-acylethanolamines (NAEs), including anandamide (20:4n-6 NAE), elicit a variety of biological effects through cannabinoid receptors, whereas saturated and monounsaturated NAEs are inactive. Arachidonic acid mobilization induced by treatment of intact mouse peritoneal macrophages with Ca2+ ionophore A23187 had no effect on the production of NAE or its precursor N-acylphosphatidylethanolamine (N-acyl PE). Addition of exogenous ethanolamine resulted in enhanced NAE synthesis by its N-acylation with endogenous fatty acids, but this pathway was not selective for arachidonic acid. Incorporation of (18)O from H2 (18)O-containing media into the amide carbonyls of both NAE and N-acyl PE demonstrated a rapid, constitutive turnover of both lipids.

Occurrence and postmortem generation of anandamide and other long-chain N-acylethanolamines in mammalian brain.

Long-chain N-acylethanolamines (NAEs) were assayed in pig, sheep and cow brain by gas chromatography/mass spectrometry of their tert.-butyldimethylsilyl derivatives in the presence of deuterium-labeled internal standards. Immediately after death, total NAEs ranged from about 2.7 micrograms/g wet weight (sheep, cow) to 6.5 micrograms/g wet weight (pig) and consisted almost exclusively (99%) of saturated and monounsaturated species. Anandamide (20:4n-6 NAE) comprised about 1% of total NAE in pig and cow brain, but was absent in freshly extracted sheep brain. When pig brain was analysed after 0.5, 1, 3, 4 and 23 h at ambient temperature, NAE levels were found to increase substantially over the entire time period with 20:4n-6 NAE formation exhibiting a time lag compared to that of saturated and monounsaturated NAEs.