€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****GENE***** Kutchan TM Heterologous expression of alkaloid biosynthetic genes--a review. In: Gene (1996 Nov 7) 179(1):73-81 ISSN: 0378-1119 Tetrahydrobenzylisoquinoline alkaloids comprise a diverse class of secondary metabolites with many pharmacologically active members. The biosynthesis at the enzyme level of at-least two tetrahydrobenzylisoquinoline alkaloids, the benzophenanthridine alkaloid sanguinarine in the California poppy, Eschscholtzia californica, and the bisbenzylisoquinoline alkaloid berbamunine in barberry, Berberis stolonifera, has been elucidated in detail starting from the aromatic amino acid (aa) L-tyrosine. In an initial attempt to develop alternate systems for the production of medicinally important alkaloids, one enzyme from each pathway (BBE, a covalently flavinylated enzyme of benzophenanthridine alkaloid biosynthesis and CYP80, a phenol coupling cytochrome P-450-dependent oxidase of bisbenzylisoquinoline alkaloid biosynthesis) has been purified to homogeneity, a partial aa sequence determined, and the corresponding cDNAs isolated with aid of synthetic oligos based on the aa sequences. The recombinant enzymes were actively expressed in Spotloptera frugiperda Sf9 cells using a baculovirus vector, purified and then characterized. Insect cell culture has proven to be a powerful system for the overexpression of alkaloid biosynthetic genes. Registry Numbers: EC 1.1 (Alcohol Oxidoreductases) EC 1.1.3.34 (berbamunine synthase) EC 1.5. (Oxidoreductases, N-Demethylating) EC 1.5.3.9 (reticuline oxidase) 2447-54-3 (sanguinarine) 9035-51-2 (Cytochrome P-450) €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****ARZNEIMITTEL-FORSCHUNG***** Reimeier C Schneider I Schneider W Schafer HL Elstner EF Effects of ethanolic extracts from Eschscholtzia californica and Corydalis cava on dimerization and oxidation of enkephalins. In: Arzneimittelforschung (1995 Feb) 45(2):132-6 ISSN: 0004-4172 The endogenous pentapeptides, met-enkephalin and leuenkephalin, similar to their parent structures, beta-endorphin or dynorphin, bind to opioid receptors of the nociceptive system thus provoking analgesic responses. Peroxidases and phenolases (tyrosinase, catecholase) were shown to dimerize these pentapeptides thus possibly modulating their activity and/or lifetime. Extracts from plants from the order of the Papaverales contain isoquinoline alkaloids. Since the benzoisoquinolines are known to possess sedative-hypnotic activities, the potential effects of extracts from two species from this plant group, Eschscholtzia californica (Papaveraceae) and tyrosinase-catalyzed dimerization and/or oxidation of met-enkephalin were investigated. The results of the study show that the peroxidase- catalyzed dimerization via the tyr-residues is especially inhibited by the C. cava extract. The tyrosinase-catalyzed reaction yields five different products A-E, according to their HPLC-retention times. Consisting of the 4:1 (v/v) combination of the extracts from E. californica and C. cava, Phytonoxon N (abbreviated as PN) stimulates the formation of minor products A, B and E, whereas the formation of the major products C and D is inhibited. Only products C and D exhibit properties similar to the peroxidase-derived dimer. Product A is likely to be identical to DOPA-enkephalin. Registry Numbers: EC 1.11.1. (Peroxidases) EC 1.14.18.1 (Monophenol Monooxygenase) 58569-55-4 (Enkephalin, Methionine) €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ Kleber E Schneider W Schafer HL Elstner EF Modulation of key reactions of the catecholamine metabolism by extracts from Eschscholtzia californica and Corydalis cava. In: Arzneimittelforschung (1995 Feb) 45(2):127-31 ISSN: 0004-4172 Aqueous-alcoholic extracts from Eschscholtzia californica inhibit the enzymatic degradation of catecholamines as well as the synthesis of adrenaline, whereas aqueous-ethanolic extracts from Corydalis cava enhance the chemical oxidation of adrenaline and the synthesis of melanine from dihydroxyphenylalanine (DOPA). Both extracts dramatically shorten the lag phase in the catalysis of phenolase probably due to their o-diphenol content, where the Corydalis extracts are 10 times more active than the Eschscholtzia preparations. Dopamine beta-hydroxylase and monoamine oxidase (MAO-B) are especially inhibited by Eschscholtzia extracts. Diamine oxidases are inhibited by both preparations to a similar extent. The results of this study may be interpreted as a cooperative function of the two preparations in establishing and preserving high catecholamine levels thus explaining their sedative, antidepressive and hypnotic activities. Registry Numbers: EC 1.14.17.1 (Dopamine beta-Hydroxylase) EC 1.14.18.1 (Monophenol Monooxygenase) EC 1.4.3.6 (Amine Oxidase (Copper-Containing)) EC 3.6.1.37 (Na(+)-K(+)-Exchanging ATPase) EC 4.1.1.25 (Tyrosine Decarboxylase) 51-43-4 (Epinephrine) 51-61-6 (Dopamine) €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ Schafer HL Schafer H Schneider W Elstner EF Sedative action of extract combinations of Eschscholtzia californica and Corydalis cava. In: Arzneimittelforschung (1995 Feb) 45(2):124-6 ISSN: 0004-4172 The herbal drug Phytonoxon N (abbreviated as PN) is indicated in nervousness induced insomnia, agitation and/or anxiety. It is composed of alcoholic drug extracts of the plants Corydalis cava (20%) and Eschscholtzia californica (80%). Both plants are rich in isoquinoline alkaloids derived from tyrosine metabolism. Recent research shows that they may influence the neurotransmitter metabolism. €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****FEBS LETTERS***** Weiler EW Kutchan TM Gorba T Brodschelm W Niesel U Bublitz F The Pseudomonas phytotoxin coronatine mimics octadecanoid signalling molecules of higher plants [published erratum appears in FEBS Lett 1994 Aug 1;349(2):317] In: FEBS Lett (1994 May 23) 345(1):9-13 ISSN: 0014-5793 The phytotoxic principle, coronatine, which is present in several pathovars of the plant pathogen, Pseudomonas syringae was shown to be highly active in completely different, jasmonate-selective bioassays. At nanomolar to micromolar concentrations, coronatine induced the accumulation of defense-related secondary metabolites in several plant cell cultures, induced transcript accumulation of the elicitor- responsive gene encoding the berberine bridge enzyme of Eschscholtzia californica, as well as the coiling response of Bryonia dioica tendrils. Biological activity critically depended upon the structure of coronatine, and slight modifications, such as methylation of the carboxyl moiety or reduction of the carbonyl group, rendered the molecules almost inactive. Coronafacic acid, obtained by hydrolysis of coronatine, was also nearly inactive. Coronatine did not elicit the accumulation of endogenous jasmonic acid in the systems analyzed. While coronafacic acid is similar in structure to jasmonic acid, we found coronatine to be a close structural analogue of the cyclic C18- precursor of jasmonic acid, 12-oxo-phytodienoic acid. The phytotoxic symptoms produced by coronatine can now be understood on the basis of the toxin's action as a mimic of the octadecanoid signalling molecules of higher plants. Registry Numbers: 2086-83-1 (Berberine) 62251-96-1 (coronatine) 67204-66-4 (12-oxophytodienoic acid) 6894-38-8 (jasmonic acid) €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****JOURNAL OF NATURAL PRODUCTS***** Tanahashi T Zenk MH New hydroxylated benzo[c]phenanthridine alkaloids from Eschscholtzia californica cell suspension cultures. In: J Nat Prod (1990 May-Jun) 53(3):579-86 ISSN: 0163-3864 From cell cultures of Eschscholtzia californica and their spent medium, three new benzo[c]phenanthridine alkaloids--namely 10- hydroxysanguinarine [2a], 12-hydroxychelirubine [4a], and 10- hydroxychelerythrine [7a]--and two new dihydrobenzo[c]phenanthridine alkaloids--10-hydroxydihydrosanguinarine [2b] and 12- hydroxydihydrochelirubine [4b]--together with the known constituents sanguinarine [1a], chelirubine [3a], macarpine [5a], dihydrosanguinarine [1b], dihydrochelirubine [3b], and dihydromacarpine [5b], were isolated and characterized. Structure elucidations were done by 1H nmr, decoupling experiments, and NOESY spectra. Isolated microsomes from E. californica, the site of hydroxylation activity within the cells, contained the whole set 1b to 8b of 5,6-dihydrobenzo[c]phenanthridines. A scheme for the biosynthesis of macarpine [5a] from protopine [9] via dihydrosanguinarine [1b] is presented. €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****PHARMACOLOGICAL RESEARCH COMMUNICATIONS***** Vincieri FF Celli S Mulinacci N Speroni E An approach to the study of the biological activity of Eschscholtzia californica Cham. In: Pharmacol Res Commun (1988 Dec) 20 Suppl 5:41-4 ISSN: 0031-6989 Pharmacological activities of Eschscholtzia californica Cham. are not yet well known. The aim of this work is to verify the pharmacological properties and to get a first identification of the active principles. €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****PHYTOCHEMISTRY***** Kutchan TM Bock A Dittrich H Heterologous expression of the plant proteins strictosidine synthase and berberine bridge enzyme in insect cell culture. In: Phytochemistry (1994 Jan) 35(2):353-60 ISSN: 0031-9422 The heterologous expression of cDNAs encoding the alkaloid biosynthetic enzymes, strictosidine synthase [EC 4.3.3.2] from Rauvolfia serpentina and the berberine bridge enzyme [(S)-reticuline: oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9] from Eschscholtzia californica, has been achieved in a cell culture (Sf9) of the fall army worm, Spodoptera frugiperda, using a baculovirus- based expression system. The expression resulted in the overproduction of each plant enzyme in a catalytically active form. The maximal production attained was 4 mg purified, active enzyme per litre cell culture for both the strictosidine synthase and berberine bridge enzymes. Registry Numbers: EC 1.5. (Oxidoreductases, N-Demethylating) EC 1.5.3.9 (reticuline oxidase) EC 2. (Transferases) EC 2.- (strictosidine synthetase) 2086-83-1 (Berberine) €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****PLANT PHYSIOLOGY***** Facchini PJ Penzes C Johnson AG Bull D Molecular characterization of berberine bridge enzyme genes from opium poppy. In: Plant Physiol (1996 Dec) 112(4):1669-77 ISSN: 0032-0889 In Papaver somniferum (opium poppy) and related species, (S)- reticuline serves as a branch-point intermediate in the biosynthesis of numerous isoquinoline alkaloids. The berberine bridge enzyme (BBE) ([S]-reticuline:oxygen oxidoreductase [methylene bridge forming], EC 1.5.3.9) catalyzes the stereospecific conversion of the N-methyl moiety of (S)-reticuline into the berberine bridge carbon of (S)- scoulerine and represents the first committed step in the pathway leading to the antimicrobial alkaloid sanguinarine. Three unique genomic clones (bbe1, bbe2, and bbe3) similar to a BBE cDNA from Eschscholtzia californica (California poppy) were isolated from opium poppy. Two clones (bbe2 and bbe3) contained frame-shift mutations of which bbe2 was identified as a putative, nonexpressed pseudogene by RNA blot hybridization using a gene-specific probe and by the lack of transient expression of a chimeric gene fusion between the bbe2 5' flanking region and a beta-glucuronidase reporter gene. Similarly, bbe1 was shown to be expressed in opium poppy plants and cultured cells. Genomic DNA blot-hybridization data were consistent with a limited number of bbe homologs. RNA blot hybridization showed that bbe genes are expressed in roots and stems of mature plants and in seedlings within 3 d after germination. Rapid and transient BBE mRNA accumulation also occurred after treatment with a fungal elicitor or with methyl jasmonate. However, sanguinarine was found only in roots, seedlings, and fungal elicitor-treated cell cultures. Registry Numbers: EC 1.5. (Oxidoreductases, N-Demethylating) EC 1.5.3.9 (reticuline oxidase) €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ *****PLANTA MEDICA***** Granger I Serradeil-le Gal C Augereau JM Gleye J Benzophenanthridine alkaloids isolated from Eschscholtzia californica cell suspension cultures interact with vasopressin (V1) receptors. In: Planta Med (1992 Feb) 58(1):35-8 ISSN: 0032-0943 Chelerythrine and sanguinarine, two benzophenanthridine alkaloids, have been isolated from a crude methanolic extract of Eschscholtzia californica cell suspension cultures by successive fractionations. These two molecules exhibited affinity for rat liver vasopressin V1 receptors and are competitive inhibitors of [3H]-vasopressin binding within the micromolar range (Ki). Chelerythrine and sanguinarine represent two of the first non-peptidic structures providing original chemical leads for the design of synthetic vasopressin compounds. Registry Numbers: 2447-54-3 (sanguinarine) 34316-15-9 (chelerythrine) €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ ***PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES*** Dittrich H Kutchan TM Molecular cloning, expression, and induction of berberine bridge enzyme, an enzyme essential to the formation of benzophenanthridine alkaloids in the response of plants to pathogenic attack. In: Proc Natl Acad Sci U S A (1991 Nov 15) 88(22):9969-73 ISSN: 0027-8424 The berberine bridge enzyme [(S)-reticuline: oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9] is a vesicular plant enzyme that catalyzes the formation of the berberine bridgehead carbon of (S)-scoulerine from the N-methyl carbon of (S)-reticuline in a specific, unparalleled reaction along the biosynthetic pathway that leads to benzophenanthridine alkaloids. Cytotoxic benzophenanthridine alkaloids are accumulated in certain species of Papaveraceae and Fumariaceae in response to pathogenic attack and, therefore, function as phytoalexins. The berberine bridge enzyme has been purified to homogeneity from elicited cell-suspension cultures of Eschscholtzia californica, and partial amino acid sequences have been determined. A cDNA, isolated from a Agt11 cDNA bank of elicited E. californica cell- suspension cultures, coded for an open reading frame of 538 amino acids. The first 22 amino acids constitute the putative signal peptide. The mature protein has a Mr of 57,352, excluding carbohydrate. The berberine bridge enzyme was heterologously expressed in a catalytically active form in Saccharomyces cerevisiae. Southern hybridization with genomic DNA suggests that there is only one gene for the enzyme in the E. californica genome. Hybridized RNA blots from elicited E. californica cell-suspension cultures revealed a rapid and transient increase in poly(A)+ RNA levels that preceded both the increase in enzyme activity and the accumulation of benzophenanthridine alkaloids, emphasizing the integral role of the berberine bridge enzyme in the plant response to pathogens. Registry Numbers: EC 1.5. (Oxidoreductases, N-Demethylating) EC 1.5.3.9 (reticuline oxidase) €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ Blechert S Brodschelm W Holder S Kammerer L Kutchan TM Mueller MJ Xia ZQ Zenk MH The octadecanoic pathway: signal molecules for the regulation of secondary pathways. In: Proc Natl Acad Sci U S A (1995 May 9) 92(10):4099-105 ISSN: 0027-8424 Plant defense against microbial pathogens and herbivores relies heavily on the induction of defense proteins and low molecular weight antibiotics. The signals between perception of the aggression, gene activation, and the subsequent biosynthesis of secondary compounds are assumed to be pentacylic oxylipin derivatives. The rapid, but transient, synthesis of cis-jasmonic acid was demonstrated after insect attack on a food plant and by microbial elicitor addition to plant suspension cultures. This effect is highly specific and not caused by a number of environmental stresses such as light, heavy metals, or cold or heat shock. Elicitation of Eschscholtzia cell cultures also led to a rapid alkalinization of the growth medium prior to jasmonate formation. Inhibition of this alkalinization process by the protein kinase inhibitor staurosporine also inhibited jasmonate formation. The induction of specific enzymes in the benzo[c]phenanthridine alkaloid pathway leading to the antimicrobial sanguinarine was induced to a qualitatively and quantitatively similar extent by fungal elicitor, methyl jasmonate, and its linolenic acid-derived precursor 12-oxophytodienoic acid. It is herein proposed that a second oxylipid cascade may exist in plants starting from linoleic acid via 15,16-dihydro-12-oxophytodienoic acid to 9,10-dihydrojasmonate. Experiments with synthetic trihomojasmonate demonstrated that beta-oxidation is not a prerequisite for biological activity and that 12-oxophytodienoic acid and derivatives are most likely fully active as signal transducers. Octadecanoic acid-derived compounds are essential elements in modulating the synthesis of antibiotic compounds and are thus integral to plant defense. Registry Numbers: 57-11-4 (stearic acid) 6894-38-8 (jasmonic acid) €€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€