No abstract

Leukotriene A4 (LTA4, 5S-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid) hydrolase (LTA4H)͞aminopeptidase is a bifunctional zinc metalloenzyme that catalyzes the final and rate-limiting step in the biosynthesis of leukotriene B4 (LTB4, 5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid), a classical chemoattractant and immune modulating lipid mediator. Two chemical features are key to the bioactivity of LTB4, namely, the chirality of the 12R-hydroxyl group and the cis-trans-trans geometry of the conjugated triene structure. From the crystal structure of LTA4H, a hydrophilic patch composed of Gln-134, Tyr-267, and Asp-375 was identified in a narrow and otherwise hydrophobic pocket, believed to bind LTA4. In addition, Asp-375 belongs to peptide K21, a previously characterized 21-residue active site-peptide to which LTA4 binds during suicide inactivation. In the present report we used site-directed mutagenesis and x-ray crystallography to show that Asp-375, but none of the other candidate residues, is specifically required for the epoxide hydrolase activity of LTA4H. Thus, mutation of Asp-375 leads to a selective loss of the enzyme's ability to generate LTB4 whereas the aminopeptidase activity is preserved. We propose that Asp-375, possibly assisted by Gln-134, acts as a critical determinant for the stereoselective introduction of the 12R-hydroxyl group and thus the biological activity of LTB4.

Leukotriene (LT) A 4 hydrolase is a bifunctional zinc metalloenzyme, which converts LTA 4 into the neutrophil chemoattractant LTB 4 and also exhibits an anion-dependent aminopeptidase activity. In the x-ray crystal structure of LTA 4 The leukotrienes (LTs) 1 are a class of structurally related lipid mediators involved in the development and maintenance of inflammatory and allergic reactions (1, 2). In the biosynthesis of LTs, 5-lipoxygenase converts arachidonic acid into the unstable epoxide LTA 4 . This intermediate may in turn be conjugated with glutathione to form the spasmogenic LTC 4 , or hydrolyzed into the proinflammatory lipid mediator LTB 4 , in a reaction catalyzed by LTA 4 hydrolase (LTA4H). Leukotriene B 4 is a classical chemoattractant of human neutrophils and triggers adherence and aggregation of leukocytes to vascular endothelium at only nanomolar concentrations (3). In addition, LTB 4 modulates immune responses (4, 5) and recent studies show that this lipid mediator is involved in early effector CD4 ϩ and CD8ϩ T cell recruitment to sites of inflammation (6 -8). Moreover, LTB 4 participates in the host-defense against infections (9, 10) and is a key mediator of platelet activating factorinduced lethal shock (11-13). These effects are signaled via specific, G protein-coupled receptors for LTB 4 (BLT 1 and BLT 2 ) (14, 15). The relative contributions of these two receptors to signaling and bioactivity are presently not clear.LTA4H (EC 3.3.2.6) is a bifunctional zinc metalloenzyme, exhibiting an anion-dependant aminopeptidase activity in addition to its epoxide hydrolase activity, i.e.

Background and Purpose: Treatment of elevated intracranial pressure (ICP) is central to neurocritical care, but not all patients are eligible for invasive ICP-monitoring. A promising noninvasive option is ultrasound measurement of the optic nerve sheath diameter (ONSD). However, meta-analyses of ONSD for elevated ICP show wide confidence intervals. This might be due to baseline variations, inter-rater variability, and varying measurement methods. No standardized protocol has been validated. Corrections for eyeball diameter (ED) and optic nerve diameter (OND) may compensate for baseline variations.We evaluated a protocol and compared two different measurement methods for ONSD ultrasound. Methods: Two operators, blinded to each other's measurements, measured ONSD, ED, and OND twice in 20 patients. ONSD was measured with two different methods in use: internal (ONSDint) or external (ONSDext) of the dura mater. Intra-class correlation (ICC)was calculated for inter-rater and intra-rater reliability.Results: ICCs for inter-rater reliability of ONSDext and ONSDint (95% confidence interval) were 0.96 (0.93, 0.98) and 0.88 (0.79, 0.94), respectively. ICCs for intra-rater reliability of ONSDext and ONSDint were 0.97 (0.94, 0.99) and 0.93 (0.87, 0.96), respectively.There was no significant bias or difference in intra-rater reliability between operators.Conclusions: ONSD can be measured with an excellent inter-and intra-rater reliability and low risk of inter-rater bias, when using this protocol. ONSDext yields a higher interand intra-rater reliability than ONSDint. Corrections for ED and OND can be performed reliably.