Leukotriene Biosynthesis Research Articles

T helper cell type 2 cytokines coordinately regulate immunoglobulin E-dependent cysteinyl leukotriene production by human cord blood-derived mast cells: profound induction of leukotriene C(4) synthase expression by interleukin 4.

Human mast cells (hMCs) derived in vitro from cord blood mononuclear cells exhibit stem cell factor (SCF)-dependent comitogenic responses to T helper cell type 2 (Th2) cytokines. As cysteinyl leukotriene (cys-LT) biosynthesis is a characteristic of immunoglobulin (Ig)E-activated mucosal hMCs, we speculated that Th2 cytokines might regulate eicosanoid generation by hMCs. After passive sensitization for 5 d with IgE in the presence of SCF, anti-IgE-stimulated hMCs elaborated minimal cys-LT (0.1 +/- 0.1 ng/10(6) hMCs) and abundant prostaglandin (PG)D(2) (16.2 +/- 10.3 ng/10(6) hMCs). Priming of hMCs by interleukin (IL)-4 with SCF during passive sensitization enhanced their anti-IgE-dependent histamine exocytosis and increased their generation of both cys-LT (by 27-fold) and PGD(2) (by 2. 5-fold). Although priming with IL-3 or IL-5 alone for 5 d with SCF minimally enhanced anti-IgE-mediated cys-LT generation, these cytokines induced further six- and fourfold increases, respectively, in IgE-dependent cys-LT generation when provided with IL-4 and SCF; this occurred without changes in PGD(2) generation or histamine exocytosis relative to hMCs primed with IL-4 alone. None of these cytokines, either alone or in combination, substantially altered the levels of cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), or 5-LO activating protein (FLAP) protein expression by hMCs. In contrast, IL-4 priming dramatically induced the steady-state expression of leukotriene C(4) synthase (LTC(4)S) mRNA within 6 h, and increased the expression of LTC(4)S protein and functional activity in a dose- and time-dependent manner, with plateaus at 10 ng/ml and 5 d, respectively. Priming by either IL-3 or IL-5, with or without IL-4, supported the localization of 5-LO to the nucleus of hMCs. Thus, different Th2-derived cytokines target distinct steps in the 5-LO/LTC(4)S biosynthetic pathway (induction of LTC(4)S expression and nuclear import of 5-LO, respectively), each of which is necessary for a full integrated functional response to IgE-dependent activation, thus modulating the effector phenotype of mature hMCs.

The N-terminal “β-Barrel” Domain of 5-Lipoxygenase Is Essential for Nuclear Membrane Translocation

5-Lipoxygenase is the key enzyme in the formation of leukotrienes, which are potent lipid mediators of asthma pathophysiology. This enzyme translocates to the nuclear envelope in a calcium-dependent manner for leukotriene biosynthesis. Eight green fluorescent protein (GFP)-lipoxygenase constructs, representing the major human and mouse enzymes within this family, were constructed and their cDNAs transfected into human embryonic kidney 293 cells. Of these eight lipoxygenases, only the 5-lipoxygenase was clearly nuclear localized and translocated to the nuclear envelope upon stimulation with the calcium ionophore. The N-terminal "beta -barrel" domain of 5-lipoxygenase, but not the catalytic domain, was necessary and sufficient for nuclear envelope translocation. The GFP-N-terminal 5-lipoxygenase domain translocated faster than GFP-5-lipoxygenase. beta-Barrel/catalytic domain chimeras with 12- and 15-lipoxygenase indicated that only the N-terminal domain of 5-lipoxygenase could carry out this translocation function. Mutations of iron atom binding ligands (His550 or deletion of C-terminal isoleucine) that disrupt nuclear localization do not alter translocation capacity indicating distinct determinants of nuclear localization and translocation. Moreover, data show that GFP-5-lipoxygenase beta-barrel containing constructs can translocate to the nuclear membrane whether cytoplasmic or nuclear localized. Thus, the predicted beta-barrel domain of 5-lipoxygenase may function like the C2 domain within protein kinase C and cytosolic phospholipase A(2) with unique determinants that direct its localization to the nuclear envelope.

ChemInform Abstract: Symmetrical Bis(heteroarylmethoxyphenyl)alkylcarboxylic Acids as Inhibitors of Leukotriene Biosynthesis.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Reduced by Contact

Reactive oxygen species (ROS) and the redox state of a cell have been implicated in several signal transduction cascades including those that regulate cell proliferation. Specifically, signaling by several growth factors, including epidermal growth factor (EGF), produces a transient increase in ROS. One effect of increases in ROS is the inactivation of tyrosine phosphatases. Pani et al. found that the redox status of cells in culture was different in sparse and dense cultures (sparse cultures have more ROS than dense cultures) and that this difference correlated with whether the cells exhibited contact inhibition. Dense cultures had lower levels of the active [guanosine triphosphate (GTP)-bound] form of the small GTPase Rac-1. Treatment of sparse cultures with drugs to inhibit leukotriene biosynthesis decreased the levels of ROS to those measured in dense cultures, suggesting that Rac-1 may stimulate leukotriene biosynthesis as a mechanism to increase ROS in sparsely cultured cells. The authors also used several other drugs to regulate tyrosine phosphatase activity (sodium orthovanadate) and redox status (dithiothreitol) of the cells; however, none of the agents used were very specific. With that caveat, the results were consistent with the hypothesis that dense cells have lower levels of ROS, which allow increased activity of tyrosine phosphatases, thereby attenuating the ability of growth factors to stimulate proliferation. Thus, the regulation of the levels of ROS through regulation of the activity of proteins such as Rac-1 may be one mechanism by which the inhibition of cell proliferation in response to cell-cell contact occurs. Pani, G., Colavitti, R., Bedogni, B., Anzevino, R., Borrello, S., and Galeotti, T. (2000) A redox signaling mechanism for density-dependent inhibition of cell growth. J. Biol. Chem. 275 : 38891-38899. [Abstract] [Full Text]

Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences

5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4 mol of FSBA/mol of 5LO (of which ATP competed with 1 mol/mol) or 0.94 mol of 2-azido-ATP/mol of 5LO (of which ATP competed with 0.77 mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleotide-binding site rather than a strict ATP-binding site. Ca(2+), which also stimulates 5LO activity, had no effect on the labelling of the nucleotide-binding site. Digestion with trypsin and peptide sequencing showed that two fragments of 5LO were labelled by 2-azido-ATP. These fragments correspond to residues 73-83 (KYWLNDDWYLK, in single-letter amino acid code) and 193-209 (FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptides were modified by the labelling, suggesting that they were immediately adjacent to the C-2 position of the adenine ring of ATP. Given the stoichiometry of the labelling, the two peptide sequences of 5LO were probably near each other in the enzyme's tertiary structure, composing or surrounding the ATP-binding site of 5LO.

Symmetrical bis(heteroarylmethoxyphenyl)alkylcarboxylic acids as inhibitors of leukotriene biosynthesis.

Symmetrical bis(quinolylmethoxyphenyl)alkylcarboxylic acids were investigated as inhibitors of leukotriene biosynthesis and 4, 4-bis(4-(2-quinolylmethoxy)phenyl)pentanoic acid sodium salt (47.Na) met our design parameters for a drug candidate (ABT-080). This compound was readily synthesized in three steps from commercially available diphenolic acid. Against intact human neutrophils, 47.Na inhibited ionophore-stimulated LTB(4) formation with an IC(50) = 20 nM. In zymosan-stimulated mouse peritoneal macrophages producing both LTC(4) and PGE(2), 47.Na showed 9000-fold selectivity for inhibition of LTC(4) (IC(50) = 0.16 nM) over PGE(2) (IC(50) = 1500 nM). Preliminary pharmacokinetic evaluation in rat and cynomolgus monkey demonstrated good oral bioavailability and elimination half-lives of 9 and 5 h, respectively. Pharmacological evaluation of leukotriene inhibition with oral dosing was demonstrated in a rat pleural inflammation model (ED(50) = 3 mg/kg) and a rat peritoneal passive anaphylaxis model (LTB(4), ED(50) = 2.5 mg/kg; LTE(4), ED(50) = 1.0 mg/kg). In a model of airway constriction induced by antigen challenge in actively sensitized guinea pigs, 47.Na dosed orally blocked bronchoconstriction with an ED(50) = 0.4 mg/kg, the most potent activity we have observed for any leukotriene inhibitor in this model. The mode of inhibitory action of 47.Na occurs at the stage of 5-lipoxygenase biosynthesis as it blocks both leukotriene pathways leading to LTB(4) and LTC(4) but not PGH(2) biosynthesis. However, 47.Na does not inhibit 5-lipoxygenase catalysis in a broken cell enzyme assay; therefore it is likely that 47.Na acts as a FLAP inhibitor.

Workup-Dependent Formation of 5-Lipoxygenase Inhibitory Boswellic Acid Analogues

Pentacyclic triterpenes from the 11-keto-boswellic acid series were identified as the active principal ingredients of Boswellia resin, inhibiting the key enzyme of leukotriene biosynthesis, 5-lipoxygenase (5-LO). Of the genuine boswellic acids hitherto characterized, 3-O-acetyl-11-keto-beta-boswellic acid, AKBA (1), proved to be the most potent inhibitor of 5-LO. In the course of purification of further boswellic acid derivatives from Boswellia resin, we observed the degradation of the natural compound 3-O-acetyl-11-hydroxy-beta-boswellic acid (2) to the thermodynamically more stable product 3-O-acetyl-9, 11-dehydro-beta-boswellic acid (4). The metastable intermediate of this conversion, under moderate conditions of workup in methanolic solutions, was identified as 3-O-acetyl-11-methoxy-beta-boswellic acid (3). The novel artifactual boswellic acid derivatives inhibited 5-LO product formation in intact cells with different characteristics: 4 almost totally abolished 5-LO activity, with an IC(50) of 0.75 microM, whereas 3 and 9,11-dehydro-beta-boswellic acid (5), the deacetylated analogue of 4, were incomplete inhibitors. The data suggest that the conditions chosen for the workup of Boswellia extracts could significantly influence the potency of their biological actions and their potential therapeutic effectiveness.

Covalent Binding of LTA4 to Nucleosides and Nucleotides

Leukotriene A4 (LTA4) is a chemically reactive conjugated triene epoxide that is formed by 5-lipoxygenase and is an intermediate in the formation of the biologically active eicosanoids leukotriene B4 and leukotriene C4. The present study was undertaken to determine whether or not LTA4 could serve as an electrophilic species that nucleosides and nucleotides could attack, ultimately resulting in a covalent adduct. Electrospray ionization mass spectrometry and tandem mass spectrometry were used to study the covalent binding of LTA4 with uridine, cytidine, adenosine, and guanosine. The reaction with guanosine was found to yield five major and at least six minor adduct species. Reversed phase HPLC and mass spectrometric data suggested that the guanosine attacked LTA4 either at carbon-12 or carbon-6 with opening the epoxide at carbon-5 to yield a series of adducts characterized by the molecular anion [M–H]− at m/z 600.3. Reactions of LTA4 with mixtures of nucleosides and nucleotides revealed that guanine-containing nucleosides were the most reactive toward LTA4. The facility of the reaction of guanine with LTA4 raises the possibility that this intermediate of leukotriene biosynthesis formed on or near the cellular nuclear envelope may react with nucleosides and nucleotides present in RNA or DNA.

Medicinal plants from Nepal: evaluation as inhibitors of leukotriene biosynthesis

The methanolic extracts of 25 different Nepalese medicinal plants were tested for their activity to inhibit the biosynthesis of leukotriene B 4 in bovine polymorphonuclear leukocytes. The selected indigenous plants are used in traditional herb remedies to treat inflammatory diseases such as asthma, bronchitis, rheumatism, and skin disorders presumed to be mediated by leukotrienes. The leaves of Zanthoxylum nepalensis were shown to be the most potent inhibitor with an IC 50 value of 11 μg/ml. The extracts obtained from Astercantha longifolia and Hedychium ellipticum also exhibited potent inhibitory action with IC 50 values of 20 and 22 μg/ml, respectively.

Arachidonic acid metabolism in inflammatory cells of patients with bronchial asthma.

Over the last few years, the demonstration of beneficial effects of leukotriene receptor antagonists in various forms of asthma has renewed clinical and pharmacologic interest in this class of lipid mediators. Several studies demonstrated an increased biosynthesis of cysteinyl leukotrienes (CysLT) in asthmatic patients. However, the reasons for the dysregulated production of CysLTs in asthmatic patients are not completely defined. An improved method of lipid mediator detection and the availability of cells isolated from human airways (by bronchoalveolar lavage [BAL] and bronchial biopsies) have allowed initial studies to address this issue. Eosinophils retrieved from inflamed airways of asthmatics have a larger arachidonic acid (AA) content than their blood counterpart. The high level of AA in these cells is primarily due to a remodeling of endogenous arachidonate pools with the accumulation of this fatty acid in a triglyceride-associated pool. In addition, elevated levels of a secretory form of phospholipase A2, the key enzyme initiating the cascade of CysLTs, are found in the BAL of asthmatics. Finally, eosinophils isolated from the BAL of asthmatics have an increased expression of LTC4 synthase. The level of expression of this enzyme correlates with the increased amount of CysLTs produced in the airways of these patients. Taken together, these data identify at least two possible mechanisms to explain the excessive CysLT production in asthmatics: 1) an increased content of AA in the glycerolipid pools of inflammatory cells 2) an enhanced activity of key biosynthetic enzymes involved in CysLT synthesis.

5-lipoxygenase is phosphorylated by p38 kinase-dependent MAPKAP kinases.

5-lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes, a group of inflammatory mediators derived from arachidonic acid (AA). Here we describe that activation of p38 mitogen-activated protein kinase in human polymorphonuclear leukocytes and in Mono Mac 6 cells leads to activation of downstream kinases, which can subsequently phosphorylate 5-LO in vitro. Different agents activated the 5-LO kinase activities, including stimuli for cellular leukotriene biosynthesis (A23187, thapsigargin, N-formyl-leucyl-phenylalanine), compounds that up-regulate the capacity for leukotriene biosynthesis (phorbol 12-myristate 13-acetate, tumor necrosis factor alpha, granulocyte/macrophage colony-stimulating factor), and well known p38 stimuli as sodium arsenite and sorbitol. For all stimuli, 5-LO kinase activation was counteracted by SB203580 (3 microM or less), an inhibitor of p38 kinase. At least two p38-dependent 5-LO kinase activities were found. Based on migration properties in in-gel kinase assays and immunoreactivity, one of these was identified as mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2). The other appeared to be MAPKAP kinase 3; however, it could not be excluded that also other p38-dependent kinases contributed. When polymorphonuclear leukocytes were incubated with sodium arsenite (strong activator of 5-LO kinases), platelet-activating factor and exogenous AA, there was a 4-fold increase in 5-LO activity as compared with incubations with only platelet-activating factor and AA. This indicates that 5-LO phosphorylation can be one factor determining cellular 5-LO activity.

15-Lipoxygenation of leukotriene A 4: Studies of 12- and 15-lipoxygenase efficiency to catalyze lipoxin formation

The unstable epoxide leukotriene (LT) A 4 is a key intermediate in leukotriene biosynthesis, but may also be transformed to lipoxins via a second lipoxygenation at C-15. The capacity of various 12- and 15-lipoxygenases, including porcine leukocyte 12-lipoxygenase, a human recombinant platelet 12-lipoxygenase preparation, human platelet cytosolic fraction, rabbit reticulocyte 15-lipoxygenase, soybean 15-lipoxygenase and human eosinophil cytosolic fraction, to catalyze conversion of LTA 4 to lipoxins was investigated and standardized against the ability of the enzymes to transform arachidonic acid to 12- or 15-hydroxyeicosatetraenoic acids (HETE), respectively. The highest ratio between the capacity to produce lipoxins and HETE (LX/HETE ratio) was obtained for porcine leukocyte 12-lipoxygenase with an LX/HETE ratio of 0.3. In addition, the human platelet 100 000× g supernatant 12-lipoxygenase preparation and the human platelet recombinant 12-lipoxygenase and human eosinophil 100 000× g supernatant 15-lipoxygenase preparation possessed considerable capacity to produce lipoxins (ratio 0.07, 0.01 and 0.02 respectively). In contrast, lipoxin formation by the rabbit reticulocyte and soybean 15-lipoxygenases was much less pronounced (LX/HETE ratios <0.002). Kinetic studies of the human lipoxygenases revealed lower apparent K m for LTA 4 (9–27 μM), as compared to the other lipoxygenases tested (58–83 μM). The recombinant human 12-lipoxygenase demonstrated the lowest K m value for LTA 4 (9 μM) whereas the porcine leukocyte 12-lipoxygenase had the highest V max. The profile of products was identical, irrespective of the lipoxygenase used. Thus, LXA 4 and 6 S-LXA 4 together with the all- trans LXA 4 and LXB 4 isomers were isolated. Production of LXB 4 was not observed with any of the lipoxygenases. The lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate was considerably more efficient to inhibit conversion of LTA 4 to lipoxins, as compared to the inhibitory effect on 12-HETE formation from arachidonic acid (IC 50 1 and 50 μM, respectively) in the human platelet cytosolic fraction.

Monoclonal anti-CD18 antibody prevents transcellular biosynthesis of cysteinyl leukotrienes in vitro and in vivo and protects against leukotriene-dependent increase in coronary vascular resistance and myocardial stiffness.

Cysteinyl leukotrienes (cys-LT) can constrict small and large vessels and increase vascular permeability. Formation of cys-LT arising from polymorphonuclear leukocytes (PMNL) and endothelial cell cooperation (transcellular synthesis) led to the hypothesis that PMNL-endothelial cell adhesion may represent a key step toward the formation of vasoactive cys-LT. We studied the effect of pretreatment with a monoclonal antibody directed against the CD18 subunit of PMNL beta(2)-integrin on the synthesis of cys-LT in a PMNL-perfused isolated rabbit heart in vitro and in a model of permanent ligature of the left descending coronary artery in the rabbit in vivo. Challenge of PMNL-perfused rabbit hearts with formyl-met-leu-phe (0.3 micromol/L) caused synthesis of cys-LT and increase in coronary perfusion pressure that were prevented by the anti-CD18 antibody. Similar results were obtained with the use of A-23187 (0.5 micromol/L) as a challenge. Persistence of PMNL-associated myeloperoxidase activity in the perfusion buffer was observed in the presence of the anti-CD18 antibody, indicating decreased PMNL infiltration. Coronary artery ligature in vivo increased urinary excretion of leukotriene E(4), supporting the activation of the 5-lipoxygenase pathway during experimental acute myocardial infarction. Pretreatment with the anti-CD18 antibody (1 mg/kg) prevented the increase in leukotriene E(4) excretion. These data support the importance of adhesion in promoting cys-LT formation, originating from PMNL-endothelial cell cooperation, and contributing to myocardial stiffness and increased coronary resistance.

Cell-specific Transcription of Leukotriene C4Synthase Involves a Kruppel-like Transcription Factor and Sp1

Leukotriene C(4) synthase (LTC(4)S) is responsible for the biosynthesis of cysteinyl leukotrienes that participate in allergic and asthmatic inflammation. We analyzed 2.1 kilobases of the 5'-flanking region of the human LTC(4)S gene, which contains three DNase I hypersensitivity sites, for its transcriptional activity when fused to a promoterless and enhancerless luciferase gene. Deletion analysis revealed a nonspecific basal promoter region between nucleotides -122 and -56 upstream of the translation start site which contains a consensus Sp1 binding site and a putative initiator element (Inr) and cell-specific enhancer regions further upstream. A single mutation of either the Sp1 binding site between nucleotides -120 and -115 or the Inr (CAGAC) between nucleotides -66 and -62 reduced the expression of the reporter gene by approximately 60%, whereas double mutations decreased the expression by approximately 80%. The incubation of nuclear extracts from THP-1 and K562 cells with a (32)P-labeled oligonucleotide containing the Sp1 site or the Inr sequence gave gel-shifted complexes that were blocked by their respective cold oligonucleotides, and antisera specific for Sp1 and Sp3 provided supershifts for the former. Linker-scanning mutations of a cell-specific regulatory region revealed that mutations from nucleotides -165 to -125 reduced reporter activity. This region contains a tandem CACCC repeat (at nucleotides -149 to -145 and -139 to -135). An oligonucleotide containing the distal CACCC motif was gel shifted by THP-1 cell nuclear extract and was supershifted by antisera to Sp1 and Sp3. Cotransfection of an Sp1 expression plasmid into Drosophila SL2 cells with a -228 to -3 LTC(4)S reporter construct transactivated the reporter gene, whereas mutations at the CACCC repeat region reduced Sp1 transactivation by approximately 66%. Similarly, the Kruppel-like factor Zf9/CPBP (core promoter-binding protein) transactivated the -228 construct in COS cells but not its CACCC mutant construct. These findings indicate the involvement of Sp1 and an Inr in non-cell-specific regulation and a Kruppel-like transcription factor and Sp1 in the cell-specific regulation of the LTC(4)S gene. These are the first such analyses of a member of a newly recognized superfamily of membrane-associated proteins involved in eicosanoid and glutathione metabolism, which contains key proteins involved in the generation of both prostanoids and cysteinyl leukotrienes.

Leukotrienes in gynaecology: the hypothetical value of anti-leukotriene therapy in dysmenorrhoea and endometriosis.

The lipoxygenase products (leukotrienes) have been demonstrated in many mammalian tissues including humans. They are widely distributed in the lungs, gut, uterus, kidneys, skin, heart and the liver. Their roles as mediators of inflammation have made them therapeutic targets. Significant amounts of leukotrienes have been demonstrated in the endometrium of women with primary dysmenorrhoea who do not respond to treatment with anti-prostaglandins. Also, in endometriosis, cytokines, which can initiate the cascade for the biosynthesis of leukotrienes, have been shown to be elevated. It is estimated that 10-30% of patients with painful periods fail to respond to prostaglandin (PG) synthetase inhibitors. Of adult females approximately 40% have painful menstruation and 10% of these are incapacitated for 1-3 days per month, and approximately 10% of women aged between 15-45 years suffer from endometriosis, which is a significant cause of infertility. Leukotriene receptor antagonists have recently been licensed for the treatment of asthma in the UK. In this review, we present the case for the potential use of these products in the management of primary dysmenorrhoea (especially in patients who are not responding to the traditional treatment using PG synthetase inhibitors) and possibly also in cases of endometriosis.

Concentration-dependent potentiating and inhibitory effects of Boswellia extracts on 5-lipoxygenase product formation in stimulated PMNL.

Preparations from the gum of Boswellia spec. have been used in the traditional medicine for the treatment of inflammatory diseases. Extracts from B. serrata gum were shown to inhibit leukotriene biosynthesis by impairing the 5-lipoxygenase (5-LO) activity. In order to identify the minimal effective concentrations of extracts in vitro we studied the effects of ethanolic extracts from commercially available resins from two regions (B. serrata gum from India and Olibanum in granis from Arabia) on the 5-LO product formation from endogenous substrate in calcium and ionophore stimulated neutrophils in a defined concentration range. Both extracts inhibited 5-LO product formation in vitro in concentrations greater than 10 to 15 micrograms/ml as reported previously for an ethanolic B. serrata extract. In contrast, lower concentrations of extracts (1 to 10 micrograms/ml) even potentiated 5-LO product formation, especially the biosynthesis of 5(S)-HETE. The in vitro data underline the major importance of drug standardization when Boswellia resin containing preparations are used for the treatment of diseases.

Four Novel Oligostilbenes from the Roots of Vitis amurensis

Two novel resveratrol trimers, amurensins C ( 1), D ( 2), and two novel resveratrol pentamers, amurensins E ( 3), F ( 4) were isolated from the roots of Vitis amurensis Rupr. Their structures and relative or absolute configurations were determined by means of spectroscopic evidence, especially HMBC and NOE experiments. 2, 3 and 4 are new types of resveratrol oligomers and are reported here for the first time. The pharmacological activities on anti-inflammation of all amurensins have been tested. Among them, compound 4 showed potent inhibition on biosynthesis of leukotriene B 4 (LTB 4).

Adenosine, a Potent Natural Suppressor of Arachidonic Acid Release and Leukotriene Biosynthesis in Human Neutrophils

Leukotrienes (LTs) constitute a family of arachidonic acid (AA) metabolites with potent biological activities. Leukotriene B 4 and the cysteinyl-LTs (LTC 4 , -D 4 , and -E 4 ) have now been implicated in a number of physiological processes and inflammatory and allergic diseases (1). The early demonstration of an involvement of the cysteinyl-LTs as mediators of the bronchospasm in asthma has provided the impetus for the development of LT synthesis inhibitors and LT antagonists, and stimulated intensive research toward the understanding of the mechanisms of LT synthesis. While our knowledge of the biochemistry of LT synthesis and the enzymes involved has made remarkable progress, particularly with the identification and cloning of the proteins of the 5-lipoxygenase (5-LO) pathway, much remains to be learned about the complex mechanisms of regulation of the biosynthesis of these important mediators of inflammation and allergy.

The molecular biology and regulation of 5-lipoxygenase.

5-Lipoxygenase (5LO) catalyzes the conversion of arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE), and further to the allylic epoxide 5(S)-trans-7,9-trans-11,14-cis-eicosatetrenoic acid (leukotriene A 4 , LTA 4 ). Thus, 5LO has a central role in leukotriene biosynthesis. This article covers some basic properties of 5LO. Studies regarding the 5LO gene and targeted gene disruption, and 5LO intracellular compartmentalization, are described in other parts of this supplement.

Intracellular Compartmentalization of Leukotriene Biosynthesis

Leukotrienes (LTs) are potent bioactive lipids derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid (AA) metabolism. Although they have been conventionally viewed as paracrine mediators of inflammatory disease processes such as asthma (1), more recent information suggests that they are also important participants in disease processes characterized by cellular proliferation and fibrogenesis (2-4), and that they subserve a homeostatic role in antimicrobial host defense (5). In view of the actions and importance of LTs, substantial effort has been directed at increasing our understanding of the regulation of their synthesis. An extensive body of research, reviewed elsewhere in this symposium, has identified three key factors that regulate LT synthesis: (1) the steady state levels of key proteins necessary for AA release and 5-LO metabolism; (2) posttranslational modifications (such as phosphorylation) that alter the catalytic activities of these proteins; and (3) the concentrations of small molecules (e.g., Ca 2+ , ATP, and glutathione) that serve as cofactors for certain of these proteins. One additional determinant of LT biosynthesis that has been recognized is the intracellular compartmentalization of these LT-forming proteins. This topic has become a focus of investigation in our own and other laboratories, and this article reviews the current state of knowledge in this area. Indeed, a number of unexpected findings that have emerged from these studies underscore the appropriateness of a perspective on LT biology that transcends conventional notions.