Metabolism of benzo(a)pyrene to reactive intermediate(s) via prostaglandin biosynthesis.

We have examined the oxidation of benzo (a) pyrene (BP) to electrophilic metabolites during the formation of prostaglandins (PG) and thromboxanes (TX) from arachidonic acid (AA) by guinea pig lung microsomal protein. In the presence of NADPH or AA, electrophilic metabolites of [¹⁴C]-BP were generated which were non-extractable from microsomal protein and thus assumed to be covalently bound. The total amount of BP metabolized in the presence of NADPH was 2–2 $\text{1}\text{2}$ times the amount of BP metabolized in the presence of AA. Only 4–5% of BP metabolized by the NADPH mediated mixed-function oxidase system was covalently bound, whereas 12–15% of the BP metabolized in the presence of AA was covalently bound to tissue protein and DNA. Quinones were the major metabolites produced by the AA dependent system, while dihydrodiols were the major metabolites formed by the NADPH dependent system. 7, 12-Dimethyl-benzanthracene, and 7,8-BP-dihydrodiol, but not 3 hydroxy-BP were also oxidized by PG synthetase to reactive metabolites.     

Studies on the covalent binding of an intermediate(s) in prostaglandin biosynthesis to tissue macromolecules.

We investigated the covalent binding of intermediates in prostaglandin biosynthesis to tissue macromolecules. Following incubation of [1-14C]arachidonic acid with the microsomal fraction from guinea pig lung, ram or bovine seminal vesicle, human platelets, rabbit kidney, or rat stomach fundus, the amount of covalent binding of arachidonic acid metabolites expressed as percentage of total arachidonic acid metabolized varied from tissue to tissue ranging from 3% in human platelets to 18.2% in ram seminal vesicles. In general, the thromboxane synthesizing tissues had less covalently bound metabolites than the other tissues. The amount of covalently bound metabolites was increased in the guinea pig lung microsomes when the thromboxane synthetase inhibitor, N-0164, was added to the incubation mixture. The covalent binding of arachidonic acid metabolite(s) was greatly reduced by the addition of glutathione to the incubation mixture. In addition to the covalently bound metabolites, water-soluble metabolites derived from arachidonic acid metabolism were also observed. The amount of water-soluble metabolites was small in each tissue except for the rat stomach fundus. In the rat stomach fundus the water-soluble metabolites accounted for over 50% of the total metabolites. Conditions which would tend to increase or decrease the levels of free prostaglandin endoperoxides during the incubation of arachidonic acid with the microsomes gave increased or decreased levels of covalent binding. Our data suggest that the prostaglandin endoperoxides are responsible for the covalent binding observed during prostaglandin biosynthesis. This covalent binding to tissue macromolecules may be of physiological and pathological significance.     

Covalent binding of an intermediate(s) in prostaglandin biosynthesis to guinea pig lung microsomal protein

We have investigated the possible covalent binding of intermediates in prostaglandin (PG) biosynthesis to tissue macromolecules. Following incubation of arachidonic acid -1-[14]C (AA) with guinea pig lung microsomes, radioactivity was associated with the microsomal protein which was not dissociated from the protein by exhaustive solvent extraction. Furthermore, filtration of the protein complex through a Sephadex G-25 column failed to dissociate the radioactivity from the protein. This probably indicates covalent binding of AA metabolite(s) to protein. [3]H-PGE₂, [3]H-PGF_2α, and [3]H-thromboxane B₂ (TXB₂) did not show this high affinity binding to microsomal protein. The covalent binding of AA metabolites was greatly reduced in denatured microsomes and was inhibited by the addition of glutathione (GSH) or indomethacin to the incubation mixtures. Chromatographic analysis of the water layers obtained from microsomal incubations with either [3]H-AA or [3]H-GSH suggested the presence of one or more glutathione conjugates derived from AA. These studies indicate that most likely an intermediate formed during PG synthesis from AA covalently binds to tissue macromolecules. This covalent binding may be of physiological and pathological significance.     

Epidermal growth factor stimulates prostaglandin biosynthesis by canine kidney (MDCK) cells.

Serum and/or arachidonic acid stimulated prostaglandin production by dog kidney (MDCK) cells. Epidermal growth factor (EGF) at concentrations of 10^?9 to 10^?10 M stimulated the biosynthesis of prostaglandins by MDCK cells but not that by human fibroblasts (D-550), mouse fibroblasts (3T3), transformed mouse fibroblasts (MC5-5), and rabbit aorta endothelial cells (CLO). EGF also stimulated the release of radioactivity from MDCK cells radioactively labelled with [³H]arachidonic acid.     

Leaf epidermal morphology of Zanthoxylum s.l. (Rutaceae) from China

Leaf epidermis characters in 40 of ca. 250 species of Zanthoxylum (Rutaceae) were investigated using light microscopy (LM) and scanning electron microscopy (SEM). The stomata are anomocytic and exist only on the abaxial epidermis except in Z. nitidum, which also has stomata on the adaxial surface. The epidermal cells are usually polygonal or irregular in shape, with anticlinal walls straight, arched, repand, or sinuous. Under the SEM, the inner margin of the outer stomatal rim is nearly smooth, sinuolate or erose, and the cuticular membrane of the leaf epidermis is smooth, striate, or sometimes striate to wrinkled. These data of leaf epidermis of Zanthoxylum demonstrated that there exist many common characters between subgen. Fagara and subgen. Zanthoxylum, sug-gesting a close relationship between the two subgenera. The utility of some characters in identifying some species of Zanthoxylum was also discussed.     

Stimulation of prostaglandin biosynthesis by lipoic acid.

Enzyme preparations from sheep seminal vesicles display an enhanced ability to synthesize prostaglandins, particularly prostaglandin F from polyunsaturated fatty acids if alpha-lipoic acid is present in the incubation mixture prior to the addition of fatty acid. The stimulation by lipoate is reversible, time dependent, and involves modifications of V and Km for oxygenase activity. Product studies, structure vs. activity studies, and purification data indicate that lipoate exerts it effect by a mechanism distinct from a glutathione-like metabolism of the endoperoxide linkage in prostaglandin G and prostaglandin H. In addition, product studies suggest that lipoate is not a cofactor for the endoperoxide isomerase component of prostaglandin synthetase. Purification of the endoperoxide synthesizing activity by ion-exchange chromatography and isoelectric focusing yields preparations which are more responsive to lipoate than microsomal preparations.     

Isolation and properties of enzymes involved in prostaglandin biosynthesis.

Prostaglandin (PG) endoperoxide synthetase was purified until homogeneity had been attained. The pure enzyme displays both cyclooxygenase and peroxidase activity, in accordance with the work of MIYAMOTO et al. (J. biol. Chem. 252, 2629--2636 (1976)). This enzyme therefore converts arachidonic acid into PGH2. Glutathione S-transferases, in the presence of glutathione, convert PGH2 into a mixture of PGF2alpha, PGE2 and PGD2. A new transferase in sheep lung gives mainly PGF2alpha and PGD2. Isolation and properties of these enzymes will be discussed. Finally, progress will be reported on the isolation of a soluble enzyme from various rat organs such as lung and spleen, which forms almost exclusively prostaglandin D.     

Inhibition of prostaglandin biosynthesis by sulphasalazine and its metabolites.

Sulphasalazine (SZ) inhibits prostaglandin (PG) biosynthesis in vitro with a potency comparable to that of aceylsalicylate. The metabolites of SZ, sulphapyridine and 5-aminosalicylic acid, were of considerably lower potency as inhibitors of PG biosynthesis in the synthetase preparations used. Th inhibition of prostaglandin production by SZ could at least partly account for the clinical utility of sulphasalazine in ulcerative colitis. Sulphapyridine may help to maintain inhibitory concentrations of SZ by restraining bacterial breakdown of the active drug.     

Inhibition of prostaglandin biosynthesis by 7-oxa- and 5-oxa-prostaglandin analogues (Short Communication)

Some oxaprostaglandin derivatives have been shown to inhibit prostaglandin biosynthesis from arachidonate by a particulate prostaglandin synthetase preparation. The most potent inhibitor was 5-oxaprost-13-trans-enoate, and inhibition by this compound appeared to be competitive. Certain structure-activity relationships were ascertained.     

Fenoprofen: Inhibitor of prostaglandin synthesis

Fenoprofen, an anti-inflammatory agent, is a potent inhibitor of prostaglandin synthesis at physiological substrate concentration. I₅₀'s of 1 μM to 100 μM were obtained when the concentration of arachidonate was increased from 1 μM to 82 μM. In contrast, a small change in I₅₀ was observed with indomethacin. Kinetic studies indicated that inhibition by fenoprofen was competitive while that of indomethacin was non-competitive.     

Accelerative autoactivation of prostaglandin biosynthesis by PGG2.

Cyclooxygenase catalysis is stimulated by its product, PGG₂, and by other lipid hydroperoxides. The endoperoxide, PGH₂, was not stimulatory. The results provide a direct demonstration of an essential role for lipid hydroperoxides in prostaglandin biosynthesis, and show how the biosynthetic intermediate PGG₂ has a positive accelerative effect.     

Effects of nitric oxide and nitric oxide-derived species on prostaglandin endoperoxide synthase and prostaglandin biosynthesis.

Prostaglandins and NO. are important mediators of inflammation and other physiological and pathophysiological processes. Continuous production of these molecules in chronic inflammatory conditions has been linked to development of autoimmune disorders, coronary artery disease, and cancer. There is mounting evidence for a biological relationship between prostanoid biosynthesis and NO. biosynthesis. Upon stimulation, many cells express high levels of nitric oxide synthase (NOS) and prostaglandin endoperoxide synthase (PGHS). There are reports of stimulation of prostaglandin biosynthesis in these cells by direct interaction between NO. and PGHS, but this is not universally observed. Clarification of the role of NO. in PGHS catalysis has been attempted by examining NO. interactions with purified PGHS, including binding to its heme prosthetic group, cysteines, and tyrosyl radicals. However, a clear picture of the mechanism of PGHS stimulation by NO. has not yet emerged. Available studies suggest that NO. may only be a precursor to the molecule that interacts with PGHS. Peroxynitrite (from O2.-+NO.) reacts directly with PGHS to activate prostaglandin synthesis. Furthermore, removal of O2.- from RAW 267.4 cells that produce NO. and PGHS inhibits prostaglandin biosynthesis to the same extent as NOS inhibitors. This interaction between reactive nitrogen species and PGHS may provide new approaches to the control of inflammation in acute and chronic settings.     

Methyl Jasmonate Induces Papain Inhibitor(s) in Tomato Leaves

Leaves of 18- to 24-d-old tomato (Lycopersicon esculentum) plants exposed to gaseous methyl jasmonate (MJ) for 24 h at 30[deg]C in continuous light contained high levels of soluble protein that inhibited papain. Chromatographic analysis demonstrated that the active protein had a molecular mass of 80 to 90 kD. Induction of papain inhibitor was directly related to the concentration of air-borne MJ up to a maximum of 0.1 [mu]L MJ per treatment and depended on the duration of exposure up to 18 h. Inhibitor activity in plants treated for less than 18 h increased with time after treatment. Levels remained constant for up to 4 d after treatment, after which time activity decreased. The youngest leaf, leaf 5, consistently lost activity at a faster rate than older, lower leaves. Inhibitor concentration in all leaves was reduced to minimum levels by 11 d after MJ treatment, but did not return to control levels. Treatment with MJ in the dark did induce inhibitor activity, but at a significantly lower rate. Polyclonal antibodies raised to purified potato tuber skin cysteine proteinase inhibitors (CPI) cross-reacted with the tomato inhibitor, suggesting that the tomato papain inhibitor and the potato CPI are closely related. No papain inhibitor activity was observed in extracts from wounded tomato leaves, nor was there any immunoreactivity with antibodies raised to potato tuber skin CPI.     

Inhibition of intestinal tone, motility and prostaglandin biosynthesis by 5,8,11,14-eicosatetraynoic acid (TYA)

Microgram concentrations of 5,8,11,14-eicosatetraynoic acid (TYA) inhibited the spontaneous increases in tone which develop in isolated guinea-pig ileum and also inhibited intestinal motility in anesthetized guinea-pigs. TYA failed to block contractions of the ileum induced by various agonists including PGE₂. It did, however, inhibit both the spontaneous liberation of spasmogenic substances from isolated ileum and the biosynthesis of PGE₂ from arachidonic acid. It is concluded that the inhibitory effects of TYA were exerted through inhibition of PG biosynthesis. Studies with antagonist drugs (atropine, methysergide and pyribenzamine) confirmed that the effects of intestinal PGs are, in the guinea-pig, largely exerted through a cholinergic mechanism.     

Snake venom metalloproteinases: structure, biosynthesis and function(s)

The biochemical and the pharmacological characterization of snake venoms revealed an important structural and functional polymorphism of proteins which they contain. Among them, snake venom metalloproteases (SVMPs) constitute approximatively 20 to 60% of the whole venom proteins. During the last decades, a significant progress was performed against structure studies and the biosynthesis of the SVMPs. Indeed, several metalloproteases were isolated and characterized against their structural and pharmacological properties. In this review, we report the most important properties concerning the classification, the structure of the various domains of the SVMPs as well as their biosynthesis and their activities as potential therapeutic agents.