Bengt Samuelsson

Ribonuclease activity and RNA binding of recombinant human Dicer

RNA silencing phenomena, known as post‐transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double‐stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA‐binding properties. The recombinant enzyme generated ∼21–23 nucleotide products from dsRNA. Processing of the microRNA let‐7 precursor by Dicer produced an apparently mature let‐7 RNA. Mg2+ was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP was dispensable for dsRNase activity in vitro. The Dicer·dsRNA complex formed at high KCl concentrations was catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA‐binding domain located at the C‐terminus of Dicer was demonstrated to bind dsRNA in vitro. Human Dicer expressed in mammalian cells colocalized with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve our understanding of RNA silencing and other Dicer‐related processes.

Expanding expression of the 5-lipoxygenase pathway within the arterial wall during human atherogenesis

Oxidation products of low-density lipoproteins have been suggested to promote inflammation during atherogenesis, and reticulocyte-type 15-lipoxygenase has been implicated to mediate this oxidation. In addition, the 5-lipoxygenase cascade leads to formation of leukotrienes, which exhibit strong proinflammatory activities in cardiovascular tissues. Here, we studied both lipoxygenase pathways in human atherosclerosis. The 5-lipoxygenase pathway was abundantly expressed in arterial walls of patients afflicted with various lesion stages of atherosclerosis of the aorta and of coronary and carotid arteries. 5-lipoxygenase localized to macrophages, dendritic cells, foam cells, mast cells, and neutrophilic granulocytes, and the number of 5-lipoxygenase expressing cells markedly increased in advanced lesions. By contrast, reticulocyte-type 15-lipoxygenase was expressed at levels that were several orders of magnitude lower than 5-lipoxygenase in both normal and diseased arteries, and its expression could not be related to lesion pathology. Our data support a model of atherogenesis in which 5-lipoxygenase cascade-dependent inflammatory circuits consisting of several leukocyte lineages and arterial wall cells evolve within the blood vessel wall during critical stages of lesion development. They raise the possibility that antileukotriene drugs may be an effective treatment regimen in late-stage disease.

Membrane Prostaglandin E Synthase-1: A Novel Therapeutic Target

Prostaglandin E2 (PGE2) is the most abundant prostaglandin in the human body. It has a large number of biological actions that it exerts via four types of receptors, EP1–4. PGE2 is formed from arachidonic acid by cyclooxygenase (COX-1 and COX-2)-catalyzed formation of prostaglandin H2 (PGH2) and further transformation by PGE synthases. The isomerization of the endoperoxide PGH2 to PGE2 is catalyzed by three different PGE synthases, viz. cytosolic PGE synthase (cPGES) and two membrane-bound PGE synthases, mPGES-1 and mPGES-2. Of these isomerases, cPGES and mPGES-2 are constitutive enzymes, whereas mPGES-1 is mainly an induced isomerase. cPGES uses PGH2 produced by COX-1 whereas mPGES-1 uses COX-2-derived endoperoxide. mPGES-2 can use both sources of PGH2. mPGES-1 is a member of the membrane associated proteins involved in eicosanoid and glutathione metabolism (MAPEG) superfamily. It requires glutathione as an essential cofactor for its activity. mPGES-1 is up-regulated in response to various proinflammatory stimuli with a concomitant increased expression of COX-2. The coordinate increased expression of COX-2 and mPGES-1 is reversed by glucocorticoids. Differences in the kinetics of the expression of the two enzymes suggest distinct regulatory mechanisms for their expression. Studies, mainly from disruption of the mPGES-1 gene in mice, indicate key roles of mPGES-1-generated PGE2 in female reproduction and in pathological conditions such as inflammation, pain, fever, anorexia, atherosclerosis, stroke, and tumorigenesis. These findings indicate that mPGES-1 is a potential target for the development of therapeutic agents for treatment of several diseases.

Structure of leukotriene c identification of the amino acid part

Abstract Leukotriene C, a “Slow Reacting Substance” (SRS) from mouse mast cell tumors, was earlier shown to be a derivative of 5-hydroxy-7,9,11,14-eicosatetraenoic acid with a cysteine containing substituent in thioether linkage at C-6 (Murphy, R.C., Hammarstrom, S., Samuelsson, B.: Proc. Natl. Acad. Sci. USA, 76 , 4275–4279 (1979)). The substituent has now been identified as γ-glutamylcysteinylglycine (glutathione).

Identification and biological activity of novel ω-oxidized metabolites of leukotriene B4 from human leukocytes

The leukotrienes constitute a new group of biologically active compounds derived from polyunsaturated fatty acids [ 11. Thus, arachidonic acid can be oxygenated by a lipoxygenase to SS-hydroperoxyeicosatetraenoic acid [2], which is further converted to an unstable epoxide, 5,6-oxido-7,9,11,14-eicosatetraenoic acid (leukotriene Aq, LTA4) [3,4]. This intermediate is transformed enzymatically by addition of glutathione into a ‘slow-reacting substance of anaphylaxis’ (SRS-A), LTC4 [5,6]. The biological activity of most SRS-A preparations is due to LTC4 and the two metabolites LTD4 and LTE4 [7-91. LTA4 can also be hydrolyzed enzymatically to 5S,12R-dihydroxy-6-cis,8,1O-trans,l4-cis-eicosatetraenoic acid (LTB4) [ 1 O-l 2] or non-enzymatically to isomeric 5,12and 5,6-dihydroxy-eicosatetraenoic acids [ 131. We have reported the formation of a novel dihydroxy-acid, 5S,12S-dihydroxy-6-trans,8-cis,lOtrans,l4-cis-eicosatetraenoic acid (SS,12SDHETE), in preparations of human leukocytes [ 14). This dihydroxy-acid was not formed via an epoxide intermediate, but by a double oxygenation of arachidonic acid. In addition, an w-hydroxylated metabolite, 5S,12S,20trihydroxy-6-trans,8-cis,lO-trarzs,lLF-cis-eicosatetraenoic acid (5S,12S,20-THETE), was identified. This report describes the formation of an w-hydroxylated metabolite of LTB4, 5S,12R,20-trihydroxy-6cis

Introduction of a nomenclature: Leukotrienes

,lO-trans,l4&s-eicosatetraenoic acid (20-OHLTB4) in human leukocyte preparations and further conversion of this trihydroxy acid to a dicarboxylic acid (20-COOH-LTB4). In addition, the biological activity of LTB4, 20-OH-LTB4 and 20-COOH-LTB4, on guinea pig lung strips is reported.

Activation of protein kinase C by lipoxin A and other eicosanoids. Intracellular action of oxygenation products of arachidonic acid

Abstract The structure of a slow reacting substance (SRS) from mouse mastocytoma cells was recently reported (Murphy, R.C., Hammarstrom, S. and Samuelsson, B. (1979) Proc. Natl. Acad. Sci. USA, in press). We proposed that SRS is formed from a previously described unstable epoxide intermediate in the formation of dihydroxylated arachidonic acid metabolites in leukocytes. The term leukotriene is introduced for compounds which like SRS are non-cyclized C20 carboxylic acids with one or two oxygen substituents and three conjugated double bonds.

Chapter 4 Coexistence of neuronal messengers — an overview

Arachidonic acid, linolenic acid and 14 different oxygenated fatty acid derivatives were tested as activators of human protein kinase C in vitro using histone as substrate. Lipoxin A (5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic activated the kinase in the presence of calcium at 30 fold lower concentration (1 microM) than did arachidonic acid or 1,3-dioleoylglycerol. The methyl ester of lipoxin A and the free acids of leukotriene B4 as well as two lipoxin B isomers were without effect. In contrast, linolenic acid, leukotriene C4, certain mono- and dihydroxylated eicosanoids and one lipoxin B isomer had stimulatory effects, albeit at higher concentrations. The substrate specificity of protein kinase C activated by lipoxin A proved to be different from that of the phosphatidylserine or phorbol ester activated kinase. Results of the present study suggest that arachidonic acid derived oxygenation products, in particular lipoxin A, may serve as intracellular activators of protein kinase C.

Leukotriene B4 is a complete secretagogue in human neutrophils: A kinetic analysis

Publisher Summary This chapter discusses results demonstrating that neurons often contain more than one chemical compound. The different types of coexistence situations are described, including (1) a classical transmitter and one or more peptides, (2) more than one classical transmitter, and (3) a classical transmitter, a peptide, and adenosine triphosphate (ATP). The functional significance of these histochemical findings is at present difficult to evaluate, but in studies on the peripheral nervous system evidence has been obtained that classical transmitter and peptide are coreleased and interact in a cooperative way on effector cells. In addition to enhancement, there is evidence that other types of interactions may occur—for example, the peptide may inhibit the release of the classical transmitter. Also in the central nervous system (CNS), indirect evidence is present for similar mechanisms—that is, to strengthen transmission at synaptic (or non-synaptic) sites and for the peptide inhibition of release of a classical transmitter. Multiple messengers may provide the means for increasing the capacity for information transfer in the nervous system.

Radioimmunoassay for Thromboxane B2

The interactions have been studied of leukotriene B4 (LTB4) and 20-COOH-LTB4 with human neutrophils (PMN). Kinetic studies, utilizing continuous recording techniques, showed that LTB4 activates PMN with respect to aggregation, mobilization of membrane-associated Ca2+, −˙ generation, and degranulation within seconds of exposure. Dose-response studies indicate 1) that LTB4 is much more potent than its dicar☐ylic acid derivative (20-COOH-LTB4) or its all trans-isomer, and 2) that PMN responses to these agents are largely dependent upon pretreatment of the cells with cytochalasin B. These properties were similar to those of the microbial ionophores, ionomycin and A23187. Results demonstrate that LTB4 rapidly activates PMN and indicate that LTB4 serves as a complete secretagogue. Moreover, they provide additional evidence that oxidized fatty acids activate human PMN.