Dual Inhibition of Mycobacterial Fatty Acid Biosynthesis and Degradation by 2-Alkynoic Acids

Summary2-Hexadecynoic acid and 2-octadecynoic acid have cidal activity against Mycobacterium smegmatis and Mycobacterium bovis BCG. At subinhibitory concentrations, M. smegmatis rapidly transformed [1-14C]-2-hexadecynoic acid into endogenous fatty acids and elongated them into mycolic acids. Toxic concentrations of 2-hexadecynoic acid resulted in accumulation of 3-ketohexadecanoic acid, which blocked fatty acid biosynthesis, and 3-hexadecynoic acid, an inhibitor of fatty acid degradation. The combination of these two metabolites is necessary to achieve the inhibition of M. smegmatis. We conclude that 2- and 3-hexa/octadecynoic acids inhibit mycolic acid biosynthesis, fatty acid biosynthesis, and fatty acid degradation, pathways of significant importance for mycobacteria

DHA and EPA Interaction with Raft Domains Observed With Solid-State 2H NMR Spectroscopy

poster abstractResearch continues to examine the health benefits of omega-3 polyunsaturated fatty acids (n-3 PUFA) found in fish oils. The major bioactive components are eicosapentaenoic acid (EPA, 20:5), with 20 carbons and 5 double bonds, and docosahexaenoic acid (DHA, 22:6), with 22 carbons and 6 double bonds. However, their molecular modes of action remain unclear. A suggested hypothesis is that these fatty acids are incorporated into membrane phospholipids and modify the structure and organization of lipid rafts, thus affecting cell signaling. We used solid-state 2H NMR spectroscopy to compare molecular organization in mixtures of 1-palmitoyl-2-eicosapentaenoylphosphatidylcholine (PEPC) and 1-palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC) with the raft-stabilizing molecules sphingomyelin (SM) and cholesterol. Our spectra for PEPC-d31 and PDPC-d31, analogs of PEPC and PDPC with a perdeuterated palmitoyl sn-1 chain, showed that DHA has a greater tendency than EPA to incorporate into raft-like domains enriched in SM and cholesterol. By using PSM-d31, an analog of SM with a perdeuterated N-palmitoyl chain, we now directly observe one of the raft-forming molecules and analyze the molecular order within the raft. These results will add to the growing information on how EPA and DHA differentially modify lipid domain organization in bilayers

Raft Busters: A Molecular Role for DHA in Biological Membranes?

poster abstractDietary consumption of fish oils rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as docosahexaenoic acid (DHA, 22:6), has a wide variety of health benefits. However, a complete molecular mechanism is yet to be elucidated. One model that has emerged from biochemical and imaging studies of cells postulates that n-3 PUFAs are taken up into phospholipids in the plasma membrane of cells and, due to their high disorder and aversion for cholesterol, reorganize lipid rafts. Lipid rafts are ordered domains within biological membranes which contain high amounts of sphingomyelin (SM) and cholesterol. To investigate this model, we studied lipid bilayers composed of SM, PDPC (a DHA-containing phospholipid), and cholesterol (1:1:1 mol). The molecular organization of each lipid was investigated with solid-state 2H NMR using deuterated analogs of the lipids. Spectral components assigned to ordered raft-like domains and disordered non-raft domains were resolved, from which the composition of the domains and the order within them could be determined. Most of the SM (84%) and cholesterol (88%) was found in the raft-like domain, together with a substantial amount of PDPC (70%). Despite the infiltration of PDPC there appears to be minimal effect on the order of SM or cholesterol. We speculate that PDPC molecules sequester into small groups minimizing the contact of DHA chains with cholesterol, thereby interrupting the continuity of the raft-like environment

DHA Alters Raft-like Membrane Domains as Revealed by Solid State 2H NMR Spectroscopy

poster abstractDietary omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as docosahexaenoic acid (DHA, 22:6), are correlated with the prevention of neurological and autoimmune disorders in humans. These fatty acids must be obtained from the diet, such as oil fish or fish oil supplements, as they cannot be generated within the human body. The origin of the health benefits at the molecular level is still under question. A membrane-mediated mechanism in which n-3 PUFAs are incorporated into phospholipids and modulate molecular organization is one possibility. Cellular membranes are inhomogeneous where structurally diverse lipids can exist in separate domains. Regions rich in sphingomyelin (SM) and cholesterol, commonly called lipid rafts, contain important signaling proteins. In a recent solid-state 2H nuclear magnetic resonance (2H NMR) study of a model membrane composed of 1-[2H31] palmitoyl-2-docosahexaenoyl-phosphatidylcholine (PDPC-d31), a deuterated analog of a DHA-containing phospholipid, in mixtures with SM and cholesterol, we discovered that DHA could significantly enter raft-like domains. How DHA affects the molecular organization within the raft-like domains is addressed here by observing PSM-d31, an analog of SM with a perdeuterated N-palmitoyl chain. The 2H NMR spectra for PSM-d31, in mixtures with PDPC and cholesterol, exhibit two spectral components, a larger more ordered component that we attribute to raft-like domains and a smaller less ordered component that we attribute to non-raft-like domains. On average, the order of PSM-d31 is reduced and, thus, disordering of PSM-d31 by PDPC is indicated. Our observations confirm that DHA can infiltrate rafts and affect molecular organization, which has implications for the signaling of raft and non-raft proteins. Furthermore, these results are consistent with in vivo studies showing that DHA infiltrates rafts

Lymphatic vasculature mediates macrophage reverse cholesterol transport in mice

Reverse cholesterol transport (RCT) refers to the mobilization of cholesterol on HDL particles (HDL-C) from extravascular tissues to plasma, ultimately for fecal excretion. Little is known about how HDL-C leaves peripheral tissues to reach plasma. We first used 2 models of disrupted lymphatic drainage from skin — 1 surgical and the other genetic — to quantitatively track RCT following injection of [3H]-cholesterol–loaded macrophages upstream of blocked or absent lymphatic vessels. Macrophage RCT was markedly impaired in both models, even at sites with a leaky vasculature. Inhibited RCT was downstream of cholesterol efflux from macrophages, since macrophage efflux of a fluorescent cholesterol analog (BODIPY-cholesterol) was not altered by impaired lymphatic drainage. We next addressed whether RCT was mediated by lymphatic vessels from the aortic wall by loading the aortae of donor atherosclerotic Apoe-deficient mice with [2H]6-labeled cholesterol and surgically transplanting these aortae into recipient Apoe-deficient mice that were treated with anti-VEGFR3 antibody to block lymphatic regrowth or with control antibody to allow such regrowth. [2H]-Cholesterol was retained in aortae of anti–VEGFR3-treated mice. Thus, the lymphatic vessel route is critical for RCT from multiple tissues, including the aortic wall. These results suggest that supporting lymphatic transport function may facilitate cholesterol clearance in therapies aimed at reversing atherosclerosis

Walking behaviours from the 1965–2003 American Heritage Time Use Study (AHTUS)

<p>Abstract</p> <p>Background</p> <p>The American Heritage Time Use Study (AHTUS) represents a harmonised historical data file of time use by adults, amalgamating surveys collected in 1965–66, 1975–76, 1985, 1992–94, and 2003. The objectives of time-use studies have ranged from evaluating household and other unpaid production of goods and services, to monitoring of media use, to comparing lifestyles of more and less privileged social groups, or to tracking broad shifts in social behaviour. The purpose of this paper is to describe the process and utility of identifying and compiling data from the AHTUS to describe a range of walking behaviours collected using time-use survey methods over almost 40 years in the USA.</p> <p>Methods</p> <p>This is a secondary data analysis of an existing amalgamated data set. Noting source survey-specific limitations in comparability of design, we determined age-standardized participation (and associated durations) in any walking, walking for exercise, walking for transport, walking the dog, sports/exercise (excluding walking), and all physical activity for those survey years for which sufficient relevant data details were available.</p> <p>Results</p> <p>Data processing revealed inconsistencies in instrument administration, coding various types of walking and in prompting other sport/exercise across surveys. Thus for the entire period, application of inferential statistics to determine trend for a range of walking behaviours could not be done with confidence. Focusing on the two most comparable survey years, 1985 and 2003, it appears that walking for exercise in America has increased in popularity on any given day (from 2.9 to 5.4% of adults) and accumulated duration amongst those who walk for exercise (from 30 to 45 mins/day). Dog walking has decreased in popularity over the same time period (from 9.4 to 2.6%). Associated duration amongst dog walkers was stable at 30 mins/day.</p> <p>Conclusion</p> <p>The noted and sometimes substantial differences in methods between the various survey administrations preclude stringent interpretation of these trends in walking behaviours and the use of conventional application of inferential statistics to evaluate significance of time trends. Although the AHTUS offers the most comprehensive attempt at harmonization yet undertaken with these individual time-use surveys, we found that any noted cross-time changes in walking and physical activity behaviour are not easily interpreted in terms of conventional epidemiological approaches and could be true changes, artefact related to instrument and method changes, or both. Public health utilization of the AHTUS, could be enhanced with greater attention to methodological issues known to influence estimation of physical activity behaviour in population. This could be achieved with cross-disciplinary collaboration between groups of experts in the various stages of these surveys.</p

Structural identification of oxidized acyl-phosphatidylcholines that induce platelet activation

Oxidation of low-density lipoprotein (LDL) generates proinflammatory and prothrombotic mediators that may play a crucial role in cardiovascular and inflammatory diseases. In order to study platelet-activating components of oxidized LDL 1-stearoyl-2-arachidonoyl-sn-glycero-3- phosphocholine, a representative of the major phospholipid species in LDL, the 1-acyl-phosphatidylcholines (PC), was oxidized by CuCl2 and H2O2. After separation by high-performance liquid chromatography, three compounds were detected which induced platelet shape change at low micromolar concentrations. Platelet activation by these compounds was distinct from the pathways stimulated by platelet-activating factor, lysophosphatidic acid, lyso-PC and thromboxane A(2), as evidenced by the use of specific receptor antagonists. Further analyses of the oxidized phospholipids by electrospray ionization mass spectrometry structurally identified them as 1-stearoyl-2-azelaoyl-sn-glycero-3-phosphocholine (m/z 694; SAzPC), 1-stearoyl-2-glutaroyl-snglycero-3- phosphocholine (m/z 638; SGPC), and 1-stearoyl-2-( 5-oxovaleroyl)-sn-glycero-3-phosphocholine (m/z 622; SOVPC). These observations demonstrate that novel 1-acyl-PC which had previously been found to stimulate interaction of monocytes with endothelial cells also induce platelet activation, a central step in acute thrombogenic and atherogenic processes. Copyright (C) 2005 S. Karger AG, Basel

Lysophosphatidic Acid Induces Neointima Formation Through PPARγ Activation

Neointimal lesions are characterized by accumulation of cells within the arterial wall and are a prelude to atherosclerotic disease. Here we report that a brief exposure to either alkyl ether analogs of the growth factor–like phospholipid lysophosphatidic acid (LPA), products generated during the oxidative modification of low density lipoprotein, or to unsaturated acyl forms of LPA induce progressive formation of neointima in vivo in a rat carotid artery model. This effect is completely inhibited by the peroxisome proliferator-activated receptor (PPAR)γ antagonist GW9662 and mimicked by PPARγ agonists Rosiglitazone and 1-O-hexadecyl-2-azeleoyl-phosphatidylcholine. In contrast, stearoyl-oxovaleryl phosphatidylcholine, a PPARα agonist and polypeptide epidermal growth factor, platelet-derived growth factor, and vascular endothelial growth factor failed to elicit neointima. The structure-activity relationship for neointima induction by LPA analogs in vivo is identical to that of PPARγ activation in vitro and disparate from that of LPA G protein–coupled receptor activation. Neointima-inducing LPA analogs up-regulated the CD36 scavenger receptor in vitro and in vivo and elicited dedifferentiation of cultured vascular smooth muscle cells that was prevented by GW9662. These results suggest that selected LPA analogs are important novel endogenous PPARγ ligands capable of mediating vascular remodeling and that activation of the nuclear transcription factor PPARγ is both necessary and sufficient for neointima formation by components of oxidized low density lipoprotein

Residual N effects from livestock manure inputs to soils

Organic inputs including livestock manures provide nitrogen (N) to crops beyond the year of their application. This so-called residual N effect should be taken into account when making decisions on N rates for individual fields, but also when interpreting N response trials in preparation of recommendations. This paper addresses general principles of residual N effects, gives literature-based estimates of them, and reviews to which extent residual N effects are included in recommendations and regulations in selected countries