Discovering oxysterols in plasma: a window on the metabolome.

While the proteome defines the expressed gene products, the metabolome results from reactions controlled by such gene products. Plasma represents an accessible "window" to the metabolome both in regard of availability and content. The wide range of the plasma metabolome, in terms of molecular diversity and abundance, makes its comprehensive analysis challenging. Here we demonstrate an analytical method designed to target one region of the metabolome, that is, oxysterols. Since the discovery of their biological activity as ligands to nuclear receptors there has been a reawakening of interest in oxysterols and their analysis. In addition, the oxysterols, 24S- and 27-hydroxycholesterol, are currently under investigation as potential biomarkers associated with neurodegenerative disorders such as Alzheimer's disease and multiple sclerosis; widespread analysis of these lipids in clinical studies will require the development of robust, sensitive and rapid analytical techniques. In this communication we present results of an investigation of the oxysterols content of human plasma using a newly developed high-performance liquid chromatography-mass spectrometry (HPLC-MS) method incorporating charge-tagging and high-resolution MS. The method has allowed the identification in plasma of monohydroxylated cholesterol molecules, 7alpha-, 24S-, and 27-hydroxycholesterol; the cholestenetriol 7alpha,27-dihydroxycholesterol; and 3beta-hydroxycholest-5-en-27-oic acid and its metabolite 3beta,7alpha-dihydroxycholest-5-en-27-oic acid. The methodology described is also applicable for the analysis of other sterols in plasma, that is, cholesterol, 7-dehydrocholesterol, and desmosterol, as well as cholesterol 5,6- seco-sterols and steroid hormones. Although involving derivatization, sample preparation is straightforward and chromatographic analysis rapid (17 min), while the MS method offers high sensitivity (ng/mL of sterol in plasma, or pg on-column) and specificity. The methodology is suitable for targeted metabolomic analysis of sterols, oxysterols, and steroid hormones opening a "window" to view this region of the metabolome.

Structure-activity relationship of 2-oxoamide inhibition of group IVA cytosolic phospholipase A2 and group V secreted phospholipase A2.

The Group IVA cytosolic phospholipase A2 (GIVA cPLA2) is a key provider of substrates for the production of eicosanoids and platelet-activating factor. We explored the structure-activity relationship of 2-oxoamide-based compounds and GIVA cPLA2 inhibition. The most potent inhibitors are derived from delta- and gamma-amino acid-based 2-oxoamides. The optimal side-chain moiety is a short nonpolar aliphatic chain. All of the newly developed 2-oxoamides as well as those previously described have now been tested with the human Group V secreted PLA2 (GV sPLA2) and the human Group VIA calcium-independent PLA2 (GVIA iPLA2). Only one 2-oxoamide compound had appreciable inhibition of GV sPLA2, and none of the potent GIVA cPLA2 inhibitors inhibited either GV sPLA2 or GVIA iPLA2. Two of these specific GIVA cPLA2 inhibitors were also found to have potent therapeutic effects in animal models of pain and inflammation at dosages well below the control nonsteroidal anti-inflammatory drugs.

Peptidase allergen Der p 1 initiates apoptosis of epithelial cells independently of tight junction proteolysis.

Loss of epithelial cell polarity, which can arise following disruption of tight junctions (TJs), is a precursor to the care-fully orchestrated removal of moribund cells from epithelia in apoptosis. Ordinarily, this cycle of events has minimally disruptive effects on the function of the epithelial barrier, but some agents have been identified that induce apoptosis and promote epithelial leakiness. The allergen Der p 1 is a cysteine peptidase that cleaves TJ adhesion proteins and induces apoptosis in epithelial cells. This suggests the possibility that, at least for some inducers of apoptosis, these events might be causally linked. We report here that Der p 1 induces epithelial apoptosis before outright cell detachment and that apoptosis occurs within the same time span as increased paracellular permeability in polarized epithelial monolayers. Whilst TJ-deficient BEAS-2B cells were resistant to Der p 1-induced apoptosis, the cell line 1HAEo-, which was also TJ deficient, was sensitive to Der p 1, providing evidence against TJ proteolysis as a cause of apoptosis. To provide direct evidence, we propagated cells that normally express TJs in low calcium medium that prevented intercellular junction assembly. These cells retained full susceptibility to Der p 1, indicating that Der p 1-induced apoptosis is independent from TJ proteolysis.

PPAR gamma-dependent anti-inflammatory action of rosiglitazone in human monocytes: suppression of TNF alpha secretion is not mediated by PTEN regulation.

Thiazolidinediones (TZDs) are insulin-sensitising drugs that are ligands for the nuclear receptor PPAR gamma. They have been shown to inhibit PMA-stimulated secretion of TNFalpha from human monocytes, although only at concentrations well in excess of circulating levels observed during TZD therapy, suggesting a mechanism of action independent of PPAR gamma activation. Here we show that insulin-sensitising concentrations of the TZD rosiglitazone partially inhibit serum- or LPS- (but not PMA-) stimulated TNF alpha secretion from primary human monocytes, with an IC(50) of around 50nM. We also show that the observed effects are independent of PPAR gamma-mediated regulation of the lipid phosphatase PTEN. Reversed stimulus specificity, IC(50) in the insulin-sensitising range, and the fact that partial inhibition of TNF alpha secretion is also observed with a structurally unrelated PPAR gamma agonist, GW7845, demonstrate a mechanism of action distinct from that observed with higher TZD concentrations. These findings thus represent the first report of a PPAR gamma-dependent and therapeutically relevant anti-inflammatory action of TZDs in isolated human monocytes.

The crystal structure of the H48Q active site mutant of human group IIA secreted phospholipase A2 at 1.5 A resolution provides an insight into the catalytic mechanism.

The human group IIA secreted PLA(2) is a 14 kDa calcium-dependent extracellular enzyme that has been characterized as an acute phase protein with important antimicrobial activity and has been implicated in signal transduction. The selective binding of this enzyme to the phospholipid substrate interface plays a crucial role in its physiological function. To study interfacial binding in the absence of catalysis, one strategy is to produce structurally intact but catalytically inactive mutants. The active site mutants H48Q, H48N, and H48A had been prepared for the secreted PLA(2)s from bovine pancreas and bee venom and retained minimal catalytic activity while the H48Q mutant showed the maximum structural integrity. Preparation of the mutant H48Q of the human group IIA enzyme unexpectedly produced an enzyme that retained significant (2-4%) catalytic activity that was contrary to expectations in view of the accepted catalytic mechanism. In this paper it is established that the high residual activity of the H48Q mutant is genuine, not due to contamination, and can be seen under a variety of assay conditions including assays in the presence of Co(2+) and Ni(2+) in place of Ca(2+). The crystallization of the H48Q mutant, yielding diffraction data to a resolution of 1.5 A, allowed a comparison with the corresponding recombinant wild-type enzyme (N1A) that was also crystallized. This comparison revealed that all of the important features of the catalytic machinery were in place and the two structures were virtually superimposable. In particular, the catalytic calcium ion occupied an identical position in the active site of the two proteins, and the catalytic water molecule (w6) was clearly resolved in the H48Q mutant. We propose that a variation of the calcium-coordinated oxyanion ("two water") mechanism involving hydrogen bonding rather than the anticipated full proton transfer to the histidine will best explain the ability of an active site glutamine to allow significant catalytic activity.