Molecular characterization of two anthranilate synthase alpha subunit genes in <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The potent anticancer and antiviral compound camptothecin (CPT) is a monoterpene indole alkaloid produced by Camptotheca acuminata. In order to investigate the biosynthetic pathway of CPT, we studied the early indole pathway, a junction between primary and secondary metabolism, which generates tryptophan for both protein synthesis and indole alkaloid production. We cloned and characterized the alpha subunit of anthranilate synthase (ASA) from Camptotheca (designated CaASA), catalyzing the first committed reaction of the indole pathway. CaASA is encoded by a highly conserved gene family in Camptotheca. The two CaASA genes are differentially regulated. The level of CaASA2 is constitutively low in Camptotheca and was found mainly in the reproductive tissues in transgenic tobacco plants carrying the CaASA2 promoter and -glucuronidase gene fusion. CaASA1 was detected to varying degrees in all Camptotheca organs examined and transiently induced to a higher level during seedling development. The spatial and developmental regulation of CaASA1 paralleled that of the previously characterized Camptotheca gene encoding the beta subunit of tryptophan synthase as well as the accumulation of CPT. These data suggest that CaASA1, rather than CaASA2, is responsible for synthesizing precursors for CPT biosynthesis in Camptotheca and that the early indole pathway and CPT biosynthesis are coordinately regulated.     

Effects of linalool on glutamatergic system in the rat cerebral cortex

Linalool is a monoterpene compound reported to be a major component of essential oils in, various aromatic species. Several Linalool-producing species are used in traditional medical systems, includingAeolanthus suaveolens G. Dom (Labiatae) used as anticonvulsant in the Brazilian Amazon. Psychopharmacological in vivo evaluation of Linalool showed that this compound have dose-dependent marked sedative effects at the Central Nervous System, including hypnotic, anticonvulsant and hypothermic properties. The present study reports an inhibitory effect of Linalool on Glutamate binding in rat cortex. It is suggested that this neurochemical effect might be underlining Linalool psychopharmacological effects. These findings provide a rational basis for many of the traditional medical use of Linalool producing plant species.     

Effect of <Emphasis Type="Italic">Glomus</Emphasis> species on physiology and biochemistry of <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

The present study on efficacy of different Glomus species, an arbuscular mycorrhizal (AM) fungus (G. aggregatum, G. fasciculatum, G. mosseae, G. intraradices) on various growth parameters such as biomass, macro and micronutrients, chlorophyll, protein, cytokinin and alkaloid content and phosphatase activity of pink flowered Catharanthus roseus plants showed that all Glomus species except G. intraradices enhanced the chlorophyll, protein, crude alkaloid, phosphorus, sulphur, manganese and copper contents of C. roseus plants along with phosphatase activity significantly over uninoculated plants. However only G. mosseae and G. fasciculatum exhibited superior symbiotic relationship with the plant. G. mosseae was found to be the best for increasing the crude alkaloid content (8.19%) in leaf and also in increasing the quantity of important alkaloids vincristine and vinblastine.     

Phytochemical differentiation of Galanthus nivalis and Galanthus elwesii (Amaryllidaceae): A case study

The alkaloid patterns of two occasionally sympatric Galanthus nivalis and Galanthus elwesii populations were studied by GC/MS. Thirty-seven alkaloids were detected, 25 for G. nivalis and 17 for G. elwesii. Only five alkaloids were found to occur in both species. The populations of Galanthus differed in their alkaloid biosynthetic pathways. Thus, the alkaloid pattern of G. nivalis was dominated by compounds coming from a para–para′ oxidative coupling of O-methylnorbelladine. The predominant alkaloids in the roots of this species were found to belong to the lycorine and tazettine structural types; bulbs were dominated by tazettine, leaves by lycorine and flowers by haemanthamine type alkaloids. In contrast, the alkaloid pattern of G. elwesii was dominated mainly by compounds coming from an ortho–para′ oxidative coupling. The predominant alkaloids in G. elwesii roots, bulbs and leaves were those of homolycorine type, whereas the flowers accumulated mainly tyramine type compounds. The chemotaxonomical value of the alkaloids found in the studied species is discussed.     

Arthpyrone L,a New Pyridone Alkaloid from a Deep-Sea Arthrinium sp., Inhibits Proliferation of MG63 Osteosarcoma Cells by Inducing G0/G1 Arrest and Apoptosis

Fractionation of the ethanol extract of a marine fungus, Arthrinium sp., afforded a new pyridone alkaloid (arthpyrone L ( 1 )), the structure with absolute configuration of which was established by comprehensive spectroscopic analyses. In vitro cell viability assays revealed that compound 1 showed antiproliferative effects toward human A549 (lung), MG63, U2OS (bone), MCF-7 and MDA-MB-231 (breast) cancer cells. MG63 cell lines were chosen for further biological evaluations and presented apoptosis and cell cycle arrest (G0/G1 phase) upon treatment of 1 . Subsequent mechanism studies demonstrated that the growth inhibition of 1 against MG63 cells was via activation of caspase-modulated apoptotic pathway and inhibition of PI3K/Akt pathway.     

A new alkaloid from south african Conium species

Recent screening of South African Conium species for alkaloids as part of taxonomic studies has yielded γ-coniceine, coniine, methylconiine, conhydrine and a new alkaloid N-methylpseudoconhydrine. The relative stereochemistry of N-methylpseudoconhydrine was ascertained by ¹H NMR decoupling experiments. This latter alkaloid was found in significant amounts in the leaf and stem of some plants investigated and was the major alkaloid along with conhydrine in the leaf and stem of one group of high altitude plants. These plants also contained significant amounts of volatile oil, the major monoterpene being myrcene.     

Methyl jasmonate vapor increases the developmentally controlled synthesis of alkaloids in Catharanthus and Cinchona seedlings

Shortly after germination, alkaloids are rapidly synthesized in seedlings of both Catharanthus roseus L.G. Don and Cinchona ledgeriana Moens. The effect of low-level, atmospheric methyl jasmonate on this developmentally controlled process was studied. In both species, about 1 p.p.m. of methyl jasmonate vapor significantly enhanced alkaloid synthesis during germination, resulting in a doubling of alkaloid content in seedlings. Treatment with methyl jasmonate resulted in increased allocation of alkaloid precursors and in enhanced enzyme activities in alkaloid biosynthesis. The ability of methyl jasmonate to increase alkaloid biosynthesis decreased with age of the seedlings. Susceptibility of the process to methyl jasmonate was confined to a narrow time interval where the developmentally regulated onset of alkaloid synthesis occurred. When methyl jasmonate was applied at later developmental stages, its ability to enhance alkaloid content in the seedlings declined sharply.     

Biotransformation of gentiopicroside by asexual mycelia of Cordyceps sinensis

The biotransformation of gentiopicroside by asexual mycelia of Cordyceps sinensis yielded two products, one of which was proved to be a new pyridine monoterpene alkaloid. The possible mechanisms were discussed.     

Linkage analysis of a rare alkaloid present in a tetraploid potato with <Emphasis Type="Italic">Solanum chacoense</Emphasis> background

The potato genotype ND4382-19 has Solanum chacoense Bitt. in its genetic background. Foliar alkaloid analysis of it and its progeny ND5873 (ND4382-19 × Chipeta) by gas chromatography–mass spectrometry (GC–MS) showed that, in addition to the expected alkaloids (solanidine, leptinidine, and acetyl-leptinidine), there was an aglycone of another rare alkaloid. Its molecular mass and some of the m/z fragment ions were similar to leptinidine, but the major fragment ion was the m/z 150 peak of solanidine. This fragmentation pattern suggested that this alkaloid is a solanidine-based compound with mass equal to leptinidine. Leptinidine differs from solanidine by an extra –OH group, but the GC–MS fragmentation pattern of the rare compound indicated hydroxylation at a different position than the C-23 of leptinidine. The exact chemical structure is still unknown, and further analysis, such as NMR will be necessary to determine the structure. Segregation analysis of ND5873 (ND4382-19 × Chipeta) showed that presence of this rare compound segregated in a 1:1 ratio, indicating that a single gene controlled its synthesis and/or accumulation in foliar tissue. Analysis with AFLP and microsatellite markers indicated that the locus-controlling presence of this alkaloid resided on potato chromosome I, with the nearest flanking AFLP markers 0.6 and 9.4 cM apart. This rare alkaloid was present in the foliage and not detected in potato tubers. Its presence in leaves did not affect resistance/susceptibility to Colorado potato beetle.     

Camptotheca acuminata 10‐hydroxycamptothecin O‐methyltransferase: an alkaloid biosynthetic enzyme co‐opted from flavonoid metabolism

The medicinal plant Camptotheca acuminata accumulates camptothecin, 10‐hydroxycamptothecin, and 10‐methoxycamptothecin as its major bioactive monoterpene indole alkaloids. Here, we describe identification and functional characterization of 10‐hydroxycamptothecin O‐methyltransferase (Ca10OMT), a member of the Diverse subclade of class II OMTs. Ca10OMT is highly active toward both its alkaloid substrate and a wide range of flavonoids in vitro and in this way contrasts with other alkaloid OMTs in the subclade that only utilize alkaloid substrates. Ca10OMT shows a strong preference for the A‐ring 7‐OH of flavonoids, which is structurally equivalent to the 10‐OH of 10‐hydroxycamptothecin. The substrates of other alkaloid OMTs in the subclade bear little similarity to flavonoids, but the 3‐D positioning of the 7‐OH, A‐ and C‐rings of flavonoids is nearly identical to the 10‐OH, A‐ and B‐rings of 10‐hydroxycamptothecin. This structural similarity likely explains the retention of flavonoid OMT activity by Ca10OMT and also why kaempferol and quercetin aglycones are potent inhibitors of its 10‐hydroxycamptothecin activity. The catalytic promiscuity and strong inhibition of Ca10OMT by flavonoid aglycones in vitro prompted us to investigate the potential physiological roles of the enzyme in vivo. Based on its regioselectivity, kinetic parameters and absence of 7‐OMT flavonoids in vivo, we conclude that the major and likely only substrate of Ca10OMTin vivo is 10‐hydroxycamptothecin. This is likely accomplished by Ca10OMT being kept spatially separated at the tissue levels from potentially inhibitory flavonoid aglycones, and flavonoid aglycones being rapidly glycosylated to non‐inhibitory flavonoid glycosides.     

Strictosidine activation in Apocynaceae: towards a "nuclear time bomb"?

Background  

The first two enzymatic steps of monoterpene indole alkaloid (MIA) biosynthetic pathway are catalysed by strictosidine synthase (STR) that condensates tryptamine and secologanin to form strictosidine and by strictosidine β-D-glucosidase (SGD) that subsequently hydrolyses the glucose moiety of strictosidine. The resulting unstable aglycon is rapidly converted into a highly reactive dialdehyde, from which more than 2,000 MIAs are derived. Many studies were conducted to elucidate the biosynthesis and regulation of pharmacologically valuable MIAs such as vinblastine and vincristine in Catharanthus roseus or ajmaline in Rauvolfia serpentina. However, very few reports focused on the MIA physiological functions.     

Genome‐guided investigation of plant natural product biosynthesis

The medicinal plant Madagascar periwinkle, Catharanthus roseus (L.) G. Don, produces hundreds of biologically active monoterpene‐derived indole alkaloid (MIA) metabolites and is the sole source of the potent, expensive anti‐cancer compounds vinblastine and vincristine. Access to a genome sequence would enable insights into the biochemistry, control, and evolution of genes responsible for MIA biosynthesis. However, generation of a near‐complete, scaffolded genome is prohibitive to small research communities due to the expense, time, and expertise required. In this study, we generated a genome assembly for C. roseus that provides a near‐comprehensive representation of the genic space that revealed the genomic context of key points within the MIA biosynthetic pathway including physically clustered genes, tandem gene duplication, expression sub‐functionalization, and putative neo‐functionalization. The genome sequence also facilitated high resolution co‐expression analyses that revealed three distinct clusters of co‐expression within the components of the MIA pathway. Coordinated biosynthesis of precursors and intermediates throughout the pathway appear to be a feature of vinblastine/vincristine biosynthesis. The C. roseus genome also revealed localization of enzyme‐rich genic regions and transporters near known biosynthetic enzymes, highlighting how even a draft genome sequence can empower the study of high‐value specialized metabolites.     

The Alkaloids of Fungi

Examination of different species of local fungi, grown on two nutritive solutions of different composition, for alkaloid formation was investigated. The formation of alkaloids was confined to four species, namely: Geotrichum candidum, Mucor hiemalis, Aspergillus flavus and Rhizopus nigricans. A comparative study of the growth as well as the formation of alkaloids by these species and by Claviceps purpurea NRRL was carried out. Methods were also described with which the different alkaloids produced by the experimental strains were identified. Peptides as well as clavine type alkaloids were detected in all cases except with Mucor hiemalis where a compound corresponding to ergosine was the only alkaloid present.     

Tryptophan decarboxylase is encoded by two autonomously regulated genes in Camptotheca acuminata which are differentially expressed during development and stress

Camptothecin (CPT) is a valuable anti-cancer monoterpene alkaloid produced by the Chinese tree Camptotheca acuminata . Tryptophan decarboxylase (TDC) supplies tryptamine for the indole moiety of CPT and its derivatives, and is considered a key step in monoterpene indole alkaloid biosynthesis as it links primary and secondary metabolism. This report describes the isolation and characterization of tdc1 and tdc2 , two autonomously regulated TDC genes from Camptotheca . When expressed in Escherichia coli , the products of each gene could decarboxylate tryptophan, but were inactive against tyrosine, phenylalanine and 3,4-dihydroxyphenylalanine (dopa). tdc1 was developmentally regulated, having its highest expression level in the apex, young stem and bark, tissues which also contain the highest levels of CPT. Expression of tdc1 also increased during seedling development and was correlated with alkaloid accumulation during germination. tdc2 expression was induced in Camptotheca leaf discs and cell suspension cultures treated with fungal elicitor or methyl jasmonate, treatments which did not affect tdc1 expression. Unlike tdc1 , tdc2 expression was not detected in any unstressed Camptotheca tissues nor in developing seedlings. These data suggest that tdc1 may be part of a developmentally regulated chemical defense system in Camptotheca , while tdc2 serves as part of a defense system induced during pathogen challenge.     

Lycopodium Alkaloids from Huperzia serrata and Their Anti-acetylcholinesterase Activities

One new fawcettimine-type alkaloid ( 1 ), one new miscellaneous-type alkaloid ( 2 ), four new lycodine-type alkaloids ( 3 – 6 ), and eight known ones ( 7 – 14 ) were isolated from the whole plants of Huperzia serrata. Their structures and absolute configurations were elucidated based on spectroscopic data, X-ray diffraction, ECD calculation and Mosher's method. Compound 1 was a rare C₁₈N₂-type Lycopodium alkaloid, possessing serratinine skeleton with an amide side chain in C-5. The absolute configuration of the 18-OH of compounds 4 – 6 were first determined by Mosher's method. Moreover, compounds 1 – 14 were assayed anti-acetylcholinesterase effect in vitro, and compound 7 showed significant anti-acetylcholinesterase activity with an IC₅₀ value of 16.18±1.64 μM.     

Pyrindicin,a New Alkaloid from a Streptomyces Strain

In the course of our examination for the alkaloid productivities of Streptomyces strains, Streptomyces sp. NA–15 was found to produce a new alkaloid, pyrindicin, in the culture medium. The strain NA–15 was found to be a variant of Streptomyces griseoflavus and was designated as S. griseoflavus var. pyr indie us nov. var.

After the culture conditions for pyrindicin production were studied, pyrindicin was obtained as its hydrochloride (mp 145°C, decomp.) from the cultured broth. The compound was shown to possess weak antimicrobial and several pharmacological activities. The LD₅₀ of the hydrochloride (ip, in mice) was 87 mg/kg.     

Indole alkaloid biosynthesis : An investigation into leucine as a possible precursor of the monoterpene portion of vindoline

Because of the nonspecific incorporation of acetate into the monoterpenoid portion of the indole alkaloids, the amino acid leucine has been investigated as an alternative precursor. Incorporations of leucine into the alkaloids vindoline and catharanthine in Vinca rosea are found to be 0.07 and 0.02%, respectively, levels comparable with those of acetate. An equation is developed to calculate the quantity of [¹³C]leucine needed to be fed in order to obtain alkaloids with sufficient ¹³C enrichment to be analysed by ¹³C nmr spectroscopy. This equation assumes that percentage incorporations do not decrease with increasing quantities of leucine fed, an assumption found to be true. [2-¹³C]Leucine was synthesized from [2-¹³C]acetic acid in an eight-step procedure in 11.5% overall yield, and fed to Vinca rosea plants. Incorporations into vindoline obtained using both ¹⁴C and ¹³C were in reasonable agreement, and the activity was found to be spread over seven carbon atoms, none of which corresponded to the two atoms at which activity was expected. It is concluded that the leucine → mevalonate → monoterpene route does not operate in indole alkaloid biosynthesis, leaving open the question of the origin of the monoterpene portion of the alkaloids. An attempt to confirm these results by degradation of ¹⁴C-labelled alkaloids was unsuccessful, vindoline proving to be unexpectedly resistant to oxidation.     

Patterns of induced and constitutive monoterpene production in conifer needles in relation to insect herbivory

Studies were conducted to determine whether herbivore-induced synthesis of monoterpenes occurs in the needles of ponderosa pine (Pinus ponderosa Lawson), lodgepole pine (P. contorta Douglas var. latifolia Engelmann), white fir (Abies concolor Lindl. and Gordon) and Engelmann spruce [Picea engelmanii (Parry) Engelm.]. In the needles of all species except Engelmann spruce, simulated herbivory significantly induced the activity of monoterpene cyclases 4–8 days after wounding. In ponderosa pine, real herbivory by last-instar tiger moth larvae (Halisdota ingens Hy. Edwards: Lepidoptera) induced a significantly larger response (4.5-fold increase in monoterpene cyclase activity) than did simulated herbivory (2.5-fold increase). To our knowledge, this is the first report of herbivore-induced increases in monoterpene synthesis in needle tissue. Despite this increase in monoterpene synthesis, we observed no significant increase in total monoterpene pool size in wounded needles compared to controls. Large increases in the rate of monoterpene volatilization were observed in response to wounding. We conclude that the volatile losses caused by tissue damage compensate for herbivore-induced monoterpene synthesis, resulting in no change in pool size. Tiger moth larvae consume ponderosa pine needles in a pattern that begins at the tip and proceeds downward to midway along the needle, at which point they move to an undamaged needle. Constitutive monoterpene concentrations and monoterpene cyclase activities were highest in the lower half of ponderosa pine needles. The monoterpene profile also differed between the upper and lower needle halves, the lower half possessing an additional one to four monoterpene forms. We propose that the increasing gradient in monoterpene concentrations and number of monoterpenes along the needle from tip to base deters feeding beyond the midway point and provides time for the induction of increased cyclase activity and production of new monoterpenes. The induction of new monoterpene synthesis may have a role in replacing monoterpenes lost through damage-induced volatilization and preventing extreme compromise of the constitutive defense system. Received: 4 June 1997 / Accepted: 2 December 1997