Oxidized alkyl phospholipids are specific, high affinity peroxisome proliferator-activated receptor gamma ligands and agonists

Synthetic high affinity peroxisome proliferator-activated receptor (PPAR) agonists are known, but biologic ligands are of low affinity. Oxidized low density lipoprotein (oxLDL) is inflammatory and signals through PPARs. We showed, by phospholipase A(1) digestion, that PPARgamma agonists in oxLDL arise from the small pool of alkyl phosphatidylcholines in LDL. We identified an abundant oxidatively fragmented alkyl phospholipid in oxLDL, hexadecyl azelaoyl phosphatidylcholine (azPC), as a high affinity ligand and agonist for PPARgamma. [(3)H]azPC bound recombinant PPARgamma with an affinity (K(d)((app)) approximately 40 nm) that was equivalent to rosiglitazone (BRL49653), and competition with rosiglitazone showed that binding occurred in the ligand-binding pocket. azPC induced PPRE reporter gene expression, as did rosiglitazone, with a half-maximal effect at 100 nm. Overexpression of PPARalpha or PPARgamma revealed that azPC was a specific PPARgamma agonist. The scavenger receptor CD36 is encoded by a PPRE-responsive gene, and azPC enhanced expression of CD36 in primary human monocytes. We found that anti-CD36 inhibited azPC uptake, and it inhibited PPRE reporter induction. Results with a small molecule phospholipid flippase mimetic suggest azPC acts intracellularly and that cellular azPC accumulation was efficient. Thus, certain alkyl phospholipid oxidation products in oxLDL are specific, high affinity extracellular ligands and agonists for PPARgamma that induce PPAR-responsive genes.     

Lysophosphatidylcholine and lyso-PAF display PAF-like activity derived from contaminating phospholipids

Lysophosphatidylcholine is an abundant component of plasma and oxidized LDL that displays several biological activities, some of which may occur through the platelet-activating factor (PAF) receptor. We find that commercial lysophosphatidylcholine, its alkyl homolog (lyso-PAF), and PAF all induce inflammation in a murine model of pleurisy. Hydrolysis of PAF to lyso-PAF by recombinant PAF acetylhydrolase abolished this eosinophilic infiltration, implying that lyso-PAF should not have displayed inflammatory activity. Saponification of lyso-PAF or PAF acetylhydrolase treatment of lyso-PAF or lysophosphatidylcholine abolished activity; neither lysolipid should contain susceptible sn-2 residues, suggesting contaminants account for the bioactivity. Lyso-PAF and to a lesser extent lysophosphatidylcholine stimulated Ca(2+) accumulation in 293 cells stably transfected with the human PAF receptor, and this was inhibited by specific PAF receptor antagonists. Again, treatment of lyso-PAF or lysophosphatidylcholine with recombinant PAF acetylhydrolase, a nonselective phospholipase A(2), or saponification of lyso-PAF destroyed the PAF-like activity, a result incompatible with lyso-PAF or lysophosphatidylcholine being the actual agonist.We conclude that neither lyso-PAF nor lysophosphatidylcholine is a PAF receptor agonist, nor are they inflammatory by themselves. We suggest that PAF or a PAF-like mimetic accounts for inflammatory effects of lysophosphatidylcholine and lyso-PAF.     

Platelet-activating factor acetylhydrolase,and not paraoxonase-1, is the oxidized phospholipid hydrolase of high density lipoprotein particles

Paraoxonase-1 (PON1), an high density lipoprotein (HDL)-associated organophosphate triesterase, suppresses atherosclerosis in an unknown way. Purified PON1 protects lipoprotein particles from oxidative modification and hydrolyzes pro-atherogenic oxidized phospholipids and the inflammatory mediator platelet-activating factor (PAF). We find human PON1 acted as a phospholipase A(2) but not as a phospholipase C or D through cleavage of phosphodiester bonds as expected. PON1 requires divalent cations, but EDTA did not block the phospholipase A(2) activity of PON1. In contrast, a serine esterase inhibitor abolished phospholipase activity even though PON1 has no active-site serine residues. PAF acetylhydrolase, an oxidized phospholipid phospholipase A(2), is a serine esterase associated with specific HDL particles. Western blotting did not reveal detectable amounts of PAF acetylhydrolase in PON1 preparations, although very low amounts of PAF acetylhydrolase might still account for PON1 phospholipase A(2) activity. We revised the standard PON1 purification by first depleting HDL of PAF acetylhydrolase to find PON1 purified in this way no longer hydrolyzed oxidized phospholipids or PAF. Serum from a donor with an inactivating mutation in the PAF acetylhydrolase gene did not hydrolyze oxidized phospholipids or PAF, yet displayed full paraoxonase activity. We conclude that PAF acetylhydrolase is the sole phospholipase A(2) of HDL and that PON1 has no phospholipase activity toward PAF or pro-atherogenic oxidized phospholipids.     

Temporal correlation of measurements of airway hyperresponsiveness in ovalbumin-sensitized mice

Airway hyperresponsiveness, airway inflammation, and reversible airway obstruction are physiological hallmarks of asthma. These responses are increasingly being studied in murine models of antigen exposure and challenge, using whole body plethysmography to noninvasively assess airway hyperresponsiveness. This approach infrequently has been correlated with indexes of airway hyperresponsiveness measured by invasive means. Furthermore, correlation with quantitative histological data for tissue infiltration by inflammatory and immune cells, particularly in the wall of airways, during daily airway challenge is lacking. To address these uncertainties, we used C57BL/6 mice that were immunized with ovalbumin or vehicle (saline) and sensitized to aerosolized ovalbumin or vehicle 8 days later. The mice were subsequently exposed to aerosolized ovalbumin or vehicle, respectively, on days 14-22. We assessed airway hyperresponsiveness to methacholine noninvasively on days 14, 15, 18, or 22; we studied the same mice 24 h later while they were anesthetized for invasive analyses of airway hyperresponsiveness. Plasma total IgE concentration was significantly higher in the ovalbumin-treated mice compared with the vehicle-treated mice, but this did not correlate with eosinophil number. Peak airway hyperresponsiveness measured by either approach correlated early during daily antigen challenge (days 14 and 15), but this correlation was lost later during subsequent daily antigen challenges (days 18 and 22). On days 14 and 15, peak airway hyperresponsiveness correlated with transmigration of neutrophils and macrophages, but not lymphocytes, in the peribronchovascular connective tissue sheaths. This extravascular accumulation was found to be focal by three-dimensional microscopy. We conclude that, although ovalbumin treatment changed lung function in mice, correlation between noninvasive and invasive measures of peak airway hyperresponsiveness was inconsistent.     

Bioactive phospholipid oxidation products

Oxidation of phospholipids results in chain-shortened fragments and oxygenated derivatives of polyunsaturated sn-2 fatty acyl residues, generating a myriad of phospholipid products. Certain oxidation products of phosphatidylcholine bind to and activate the human receptor for PAF, and these PAF-like lipids are potent, selective inflammatory mediators. Formation of PAF-like lipids is nonenzymatic and so their accumulation is unregulated. PAF-like lipids are produced in vivo in response to oxidative stresses and are responsible for attendant acute inflammatory responses. PAF-like lipids almost exclusively contain an ether-linked alkyl residue at the sn-1 position of the phosphatidylcholine backbone and molecular identification of these is facilitated by phospholipase A₁ treatment to remove the bulk of the inactive phospholipids. The identity of biologically active species generated by oxidative fragmentation and oxidation can be elucidated by understanding relevant reactions leading to the formation of PAF-like lipids, and then their structure can be established by tandem mass spectrometry and chemical synthesis.     

Losing the desire: selection can promote obligate asexuality

Whilst parthenogenesis has evolved multiple times from sexual invertebrate and vertebrate lineages, the drivers and consequences of the sex-asex transition remain mostly uncertain. A model by Stouthamer et al. recently published in BMC Evolutionary Biology shows a pathway by which obligate asexuality could be selected for following endosymbiont infection.     

Comparing effectiveness of top-down and bottom-up strategies in containing influenza

This research compares the performance of bottom-up, self-motivated behavioral interventions with top-down interventions targeted at controlling an "Influenza-like-illness". Both types of interventions use a variant of the ring strategy. In the first case, when the fraction of a person's direct contacts who are diagnosed exceeds a threshold, that person decides to seek prophylaxis, e.g. vaccine or antivirals; in the second case, we consider two intervention protocols, denoted Block and School: when a fraction of people who are diagnosed in a Census Block (resp., School) exceeds the threshold, prophylax the entire Block (resp., School). Results show that the bottom-up strategy outperforms the top-down strategies under our parameter settings. Even in situations where the Block strategy reduces the overall attack rate well, it incurs a much higher cost. These findings lend credence to the notion that if people used antivirals effectively, making them available quickly on demand to private citizens could be a very effective way to control an outbreak.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.