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First report of field outbreaks of ergot-alkaloid toxicity in South Africa.

Outbreaks of clinical disease caused by the ingestion of ergotized Lolium rigidum (annual ryegrass), which resulted in a substantial loss in production, have been reported. A number of outbreaks of a hyperthermia syndrome in cattle, characterized by severe loss in milk production, loss of body mass and reduced fertility, are described. In one major outbreak in March to April 1994, a milling company reported that 2,646 dairy cows on 29 farms had developed clinical signs. In this outbreak, significant levels of ergotamine, ergosine, ergocornine and ergocryptine were found in the milled dairy rations fed to the affected cows. Barley screenings containing ergotized annual-ryegrass seed was identified as the toxic component and probable source of the ergot alkaloids in the ration. The clinical syndrome was reproduced experimentally by feeding suspected feed to a group of nine high-producing Ayrshire cows. An outbreak of gangrenous necrosis of the extremities in young cattle in the winter of 1987 was also suspected of having been caused by ergot alkaloids in grain screenings.

Animal Feed↗

The ergot alkaloid gene cluster in Claviceps purpurea: extension of the cluster sequence and intra species evolution.

The genomic region of Claviceps purpurea strain P1 containing the ergot alkaloid gene cluster [Tudzynski, P., Hölter, K., Correia, T., Arntz, C., Grammel, N., Keller, U., 1999. Evidence for an ergot alkaloid gene cluster in Claviceps purpurea. Mol. Gen. Genet. 261, 133-141] was explored by chromosome walking, and additional genes probably involved in the ergot alkaloid biosynthesis have been identified. The putative cluster sequence (extending over 68.5kb) contains 4 different nonribosomal peptide synthetase (NRPS) genes and several putative oxidases. Northern analysis showed that most of the genes were co-regulated (repressed by high phosphate), and identified probable flanking genes by lack of co-regulation. Comparison of the cluster sequences of strain P1, an ergotamine producer, with that of strain ECC93, an ergocristine producer, showed high conservation of most of the cluster genes, but significant variation in the NRPS modules, strongly suggesting that evolution of these chemical races of C. purpurea is determined by evolution of NRPS module specificity.

Amino Acid Sequence↗

[Pharmacological basis for the therapeutic use of ergot alkaloids].

The main mechanisms of action of ergot alkaloids are discussed. Their clinical use (migraine, uterine haemorrhage, orthostatic hypotension, senile cerebral insufficiency, hyperprolactinaemia or Parkinson's disease) and their adverse effects are explained by an affinity for three kinds of central and peripheral receptors: alpha adrenergic, serotoninergic or dopaminergic receptors.

Adrenergic alpha-Agonists↗

Physiological controls and regulation of ergot alkaloid formation.

Innovation and technical development of ergot alkaloids (EA) has moved closer to scientific research. Circumstantial evidence presently links the initiation of EA metabolism to changes in a range of parameters--morphology, concentrations of enzymes and their substrates, nutrients and external stress. The biosynthesis of EA begins at the level of the genetic information apparatus and continues at the level of physiological expression. EA and their formation play a role in the physiology of the production organism. Insufficient insight into Claviceps physiology hampers the deployment of computers in the control and regulation of the EA process. Knowledge of physiological controls and genetic manipulation are the principal tools of modern EA production. In principle it is now possible to improve EA yields by a concerted breeding of the ergot fungus by sexual and parasexual genetic engineering.

Biotechnology↗

Norleucine, a natural occurrence in a novel ergot alkaloid gamma-ergokryptinine.

A novel natural peptide ergot alkaloid gamma-ergokryptinine containing norleucine has been isolated from ergot sclerotia of the field-growing parasitic fungus Claviceps purpurea CCM 8059. Its structure was deduced from the NMR and mass spectral data. The final structural proof was provided by the crystal structure determination, which is the first X-ray structure of a natural Nle-containing secondary metabolite. The conformations of three ergopeptinines: gamma-ergokryptinine, ergoladinine, and alpha-ergokryptinine were compared.

Claviceps↗

Ergot alkaloids in cereal products: results from the Bavarian Health and Food Safety Authority.

The danger of ergot alkaloids has been well known for centuries. The origin and toxicology of these compounds have been clarified and flour mills assure us that they have mastered the problem technologically. Nevertheless, one can find food products with sometimes relatively high contents of these toxic alkaloids. Here we report on our experiences and results of an LC-MS/MS-method which we have developed to determine ergot alkaloids and which was applied to commercial products during the past two years.

Bread↗

Applications of vinylogous Mannich reactions. Total syntheses of the Ergot alkaloids rugulovasines A and B and setoclavine.

Concise syntheses of the Ergot alkaloids rugulovasine A (3a), rugulovasine B (3b), and setoclavine (2) have been completed by strategies that feature inter- and intramolecular vinylogous Mannich reactions as the key steps. Thus, the first synthesis of 3a,b commenced with the conversion of the known indole 17 into 24 via the addition of the furan 22 to the iminium ion 21, which was generated in situ from the aldehyde 19. Cyclization of 24 by a novel S(RN)1 reaction followed by removal of the N-benzyl group furnished a mixture (1:2) of 3a and 3b. In an alternative approach to these alkaloids, the biaryl 35 was reduced with DIBAL-H to give an intermediate imine that underwent spontaneous cyclization via an intramolecular vinylogous Mannich addition to provide 36a,b. N-Methylation of the derived benzyl carbamates 37a,b followed by global deprotection gave a mixture (2:1) of rugulovasines A and B (3a,b). Setoclavine (2) was then prepared from the biaryl 41 using a closely related intramolecular vinylogous Mannich reaction to furnish the spirocyclic lactones 42a,b. These lactones were subsequently transformed by hydride reduction and reductive methylation into the ergoline derivatives 43a,b, which were in turn converted into 2 by deprotection and solvolytic 1,3-rearrangement of the allylic hydroxyl group.

Alkaloids↗

Effects of topical natural ergot alkaloids on intraocular pressure and aqueous humor dynamics in ocular normotensive rabbits.

Although it has been suggested that ergot derivatives may play a role in antiglaucoma therapy, little attention has been paid to the ocular hypotensive action of these drugs. Having previously reported in our laboratory that topical dihydroergocristine decreases intraocular pressure both in ocular normotensive and alpha-chymotrypsin-induced ocular hypertensive rabbits, the aim of the present work was to assess the effect of natural ergot alkaloids, ergocristine, alpha-ergocryptine and ergocornine, on the intraocular pressure and aqueous humor dynamics in ocular normotensive rabbits in order to further explore the ocular actions of these compounds. Intraocular pressure was measured with a pneumatonometer manometrically calibrated for the rabbit eye. Changes in tonographic facility of aqueous humor outflow and rate of aqueous humor inflow were evaluated in anesthetized rabbits. Natural ergot alkaloids were found to reduce intraocular pressure in ocular normotensive eyes in a dose-related fashion. These compounds decreased both tonographic outflow facility and, to a greater extent, aqueous humor inflow, which explains their final hypotensive effect.

Administration, Topical↗

[Preservation of Claviceps purpurea, the producer of peptide ergot alkaloids, by the method of freeze drying].

A saprotrophic strain of Claviceps purpurea VNIIA 312A, an organism producing peptide +ergot alkaloids with prolactin inhibiting activity was shown to die under lyophilization conditions. To provide long-term storage of strain 312A, L-drying or drying under vacuum from liquid state was used with success. Three protective media were tested. Favourable results were obtained by using 25 per cent maltose solution as a protective medium. Preservation of the culture viability was accompanied by maintenance of the culture capacity for active formation of the biomass and production of +ergot alkaloids.

Claviceps↗

Quantitative determination of five ergot alkaloids in rye flour by liquid chromatography-electrospray ionisation tandem mass spectrometry.

A confirmatory method for detecting five ergot alkaloids, ergocristine, ergotamine, ergonovine, ergocornine and alpha-ergokryptine, in rye flour is described using high performance liquid chromatography coupled to tandem mass spectrometry detection by monitoring two transition reactions per analyte. The procedure entails a liquid-liquid extraction followed by a clean-up step using a C18 solid-phase extraction (SPE) cartridge. An analogue compound, methysergide hydrogen maleinate, was used to assess both repeatability sample preparation and potential MS response fluctuations. The method was fully validated according to the European Union (EU) criteria. Detection and quantification limits of all analytes were calculated ranging from 7 to 11 microg/kg and from 23 to 37 microg/kg, respectively. Fifteen rye flour samples were investigated with the newly developed method, and none of them were above the current Swiss limits of 200mg/kg for total ergot alkaloids.

Chromatography, High Pressure Liquid↗

The Claviceps purpurea gene encoding dimethylallyltryptophan synthase, the committed step for ergot alkaloid biosynthesis.

The first pathway-specific step of ergot alkaloid biosynthesis in the fungus, Claviceps purpurea, is catalyzed by the prenyltransferase, 4-(gamma,gamma-dimethylallyl)tryptophan synthase. Partial sequence information was obtained for the purified enzyme and a degenerate oligonucleotide mixture was used to identify and amplify segments of the gene, dmaW. The complete gene and near-full-length cDNA were cloned and sequenced. The cDNA was cloned in a yeast expression vector in sense and antisense orientations relative to the inducible GAL1 promoter. Extracts of yeast transformants with the sense constructs, but not antisense constructs or cloning vector, catalyzed production of 4-(gamma,gamma-dimethylallyl)tryptophan. The sequence of dmaW and its cDNA indicated that it encoded a 455 amino acid polypeptide with a predicted molecular mass of 51,824 Da and a putative prenyl diphosphate binding motif.

Alkyl and Aryl Transferases↗

Expression profiling of heat stress effects on mice fed ergot alkaloids.

Fescue toxicosis affects wild and domestic animals consuming ergot alkaloids contained in tall fescue forage infected with the endophytic fungus, Neotyphodium coenophialum. When animals are consuming infected fescue (E+) forage during periods of elevated ambient temperatures (summer), a range of phenotypic disorders collectively called summer slump is observed. It is characterized by hyperthermia, with an accompanying decrease in feed intake, growth, milk yield, and reproductive fitness. Laboratory mice also exhibit symptoms of fescue toxicosis at thermoneutral (TN) temperature, as indicated by reduced growth rate and reproductive fitness. Our goal was to characterize the differences in gene expression in liver of mice exposed to summer-type heat stress (HS) and E+ when compared to mice fed E+ at TN temperature. Mice were fed E+ diet under HS (34 +/- 1 degrees C; n = 13; E+HS) or TN conditions (24 +/- 1 degrees C; n = 14; E+TN) for a period of 2 weeks between 47 and 60 days of age. Genes differentially expressed between E+HS versus E+TN were identified using DNA microarrays. Forty-one genes were differentially expressed between treatment groups. Expressions of eight genes were measured using quantitative real-time PCR. Genes coding for phase I detoxification enzymes were upregulated in E+HS mouse liver. This detoxification pathway is known to produce reactive oxidative species. We observed an upregulation of genes involved in the protection against reactive oxidative species. Key genes involved in de novo lipogenesis and lipid transport were also upregulated. Finally, genes involved in DNA damage control and unfolded protein responses were downregulated.

Animal Feed↗

[Effect of the composition of a nutrient medium on growth and synthesis of ergot alkaloids by Claviceps purpurea in the saprotrophic culture].

The impact of changes in the ratio of the medium-668 components on growth and synthesis of ergot alkaloids in saprotrophic cultures was studied with mathematical design of the experiment. Medium-668 used for cultivation of strain VNIIA-312A-producing peptide +ergot alkaloids was shown to be balanced with respect to the ratio of all the medium components. An important role of phosphate in control of culture growth and alkaloids synthesis was elucidated. It was demonstrated that by changing the ratio of the medium components it was possible to control accumulation and excretion of the alkaloids which permitted development of the conditions required for product isolation.

Citrates↗

Ergot alkaloids inhibit 3H-naloxone binding to opiate receptors in the rat striatum and hippocampus.

The authors tested direct effect of selected ergot alkaloids (lisuride, terguride, DH-ergotoxine, DH-ergotamine and DH-ergocristine) on specific 3H-naloxone binding in the rat striatum and hippocampus. In the striatum they found that DH-ergotoxine (a substance with high affinity for noradrenergic receptors) inhibited specific 3H-naloxone binding much more strongly than lisuride and terguride (substances with a greater affinity for dopaminergic and serotoninergic receptors). DH-ergotoxine, which inhibited binding significantly more in the striatum than in the hippocampus, displayed the greatest activity. The results show differences in the degree of inhibition by the various groups of ergot alkaloids in the striatum. In the case of DH-ergotoxine there was also a difference in the degree of inhibition in the striatum and the hippocampus.

Animals↗

Interaction of ergot alkaloids and their combination (co-dergocrine) with alpha-adrenoceptors in the CNS.

Co-dergocrine (Hydergine), composed of four dihydrogenated peptide ergot alkaloids (dihydroergocornine, dihydroergocristine, dihydro-alpha-ergokryptine and dihydro-beta-ergokryptine), has been reported to interact with alpha-adrenoceptors. The effect of the combination and its individual components on alpha-adrenoceptors subtypes in the rat brain was investigated in the present study. All five ergot drugs displaced [3H]rauwolscine, [3H]clonidine and [3H]WB 4101 from specific binding sites in membrane preparations from rat and bovine brain at nanomolar concentrations. In rat cerebral occipital cortex slices, the ergot drugs inhibited 1-noradrenaline-stimulated cyclic AMP formation (alpha 1-adrenoceptor test) and facilitated electrically evoked noradrenaline release (alpha 2-adrenoceptor test) at nanomolar concentrations. The results from the functional tests suggest that the ergot drugs have a slightly higher affinity to alpha 2-adrenoceptors which are antagonised in a competitive manner. The alpha 1-adrenoceptors are antagonised by the ergot drugs in a non-competitive manner. The relative order of potency at both receptor types was similar in that dihydroergocornine, dihydro-alpha-ergokryptine and dihydro-beta-ergokryptine were equipotent, whereas dihydroergocristine was less potent. The effect of the combination of the ergot alkaloids at both alpha-adrenoceptors appears to reflect the summation of the contributions of its components. The differences seen in the functional tests were less pronounced in the binding tests.

Animals↗

Selective influence of ergot alkaloids on cortical and striatal dopaminergic and sergotonergic receptors.

The ergot alkaloids studied do exert selective effects on monoamine receptor systems. Lisuride acts as a very potent stimulator of adenylate cyclase in cortical brain regions, and may function as a mixed agonist-antagonist at high concentrations. It is most likely that in cortex, lisuride effects both dopamine and serotonin receptors, but predominantly serotonin receptors coupled to adenylate cyclase. The antagonist molindone exhibits selectivity for cortical serotonin-stimulated cyclase versus dopamine-stimulated cyclase and may prove useful for further elucidating the sites of lisuride action. LSD interacts with serotonin-stimulated cortical adenylate cyclase at higher concentrations than are needed for lisuride stimulation but, nevertheless, at lower concentrations than for serotonin itself (2-4). Bromocriptine, lergotrile and ergonovine may also act as agonists in stimulating adenylate cyclase, but with considerably less potency, and with differences in regional specificity for this stimulation, from lisuride and LSD. Each of these ergots may act as a mixed agonist-antagonist at high concentrations. With respect to the regions studied, antagonist effects on cyclase appear to be more prominent in striatum than in the cortical regions. The greater specificity of lisuride for serotonergic cortical receptors should make this compound useful in further studies of this system.

Adenylyl Cyclases↗