ergoline and agroclavine

Different lineages of fungi produce distinct classes of ergot alkaloids. Lysergic acid-derived ergot alkaloids produced by fungi in the Clavicipitaceae are particularly important in agriculture and medicine. The pathway to lysergic acid is partly elucidated, but the gene encoding the enzyme that oxidizes the intermediate agroclavine is unknown. We investigated two candidate agroclavine oxidase genes from the fungus Epichloë festucae var. lolii × Epichloë typhina isolate Lp1 (henceforth referred to as Epichloë sp. Lp1), which produces lysergic acid-derived ergot alkaloids. Candidate genes easH and cloA were expressed in a mutant strain of the mold Aspergillus fumigatus, which typically produces a subclass of ergot alkaloids not derived from agroclavine or lysergic acid. Candidate genes were coexpressed with the Epichloë sp. Lp1 allele of easA, which encodes an enzyme that catalyzed the synthesis of agroclavine from an A. fumigatus intermediate; the agroclavine then served as the substrate for the candidate agroclavine oxidases. Strains expressing easA and cloA from Epichloë sp. Lp1 produced lysergic acid from agroclavine, a process requiring a cumulative six-electron oxidation and a double-bond isomerization. Strains that accumulated excess agroclavine (as a result of Epichloë sp. Lp1 easA expression in the absence of cloA) metabolized it into two novel ergot alkaloids for which provisional structures were proposed on the basis of mass spectra and precursor feeding studies. Our data indicate that CloA catalyzes multiple reactions to produce lysergic acid from agroclavine and that combining genes from different ergot alkaloid pathways provides an effective strategy to engineer important pathway molecules and novel ergot alkaloids.

Topics: Aspergillus fumigatus; Epichloe; Ergolines; Ergot Alkaloids; Fungal Proteins; Industrial Microbiology; Lysergic Acid; Mass Spectrometry; Mixed Function Oxygenases; Molecular Structure; Mutation

Ergot alkaloids are indole derivatives with diverse structures and biological activities. They are produced by a wide range of fungi with Claviceps purpurea as the most important producer for medical use. Chanoclavine-I aldehyde is proposed as a branch point via festuclavine or pyroclavine to clavine-type alkaloids in Trichocomaceae and via agroclavine to ergoamides and ergopeptines in Clavicipitaceae. Here we report the conversion of chanoclavine-I aldehyde to agroclavine by EasG from Claviceps purpurea, a homologue of the festuclavine synthase FgaFS in Aspergillus fumigatus, in the presence of reduced glutathione and NADPH. EasG comprises 290 amino acids with a molecular mass of about 31.9 kDa. The soluble monomeric His(6)-EasG was purified after overproduction in E. coli by affinity chromatography and used for enzyme assays. The structure of agroclavine was unequivocally elucidated by NMR and MS analyses.

Topics: Aldehydes; Biocatalysis; Claviceps; Ergolines; Ergot Alkaloids; Fungal Proteins; Glutathione; Molecular Conformation; Molecular Sequence Data; Oxidoreductases Acting on CH-NH Group Donors; Stereoisomerism

To learn whether the low flux density combined magnetic field (CMF) can affect rats pretreated with a dopamine agonist, the frequency spectra of cortical and hippocampal EEG were studied after intraperitoneal injection of agroclavine (50 microg/kg, Day 1) either alone (N=5) or combined with 30-min exposure to CMF (B(AC)=50 microT, f(AC)=20.9Hz, B(DC)=27.3 microT) (N=5) an hour post-injection. The CMF caused an immediate and robust increase in the hippocampal beta(2) activity (22.5-30.5Hz) vs. its value in the pre-exposure period. This effect persisted during 30-min post-exposure period. After vehicle injection (0.1% ethanol) on Day 10, the beta(2) activity increased gradually during the exposure to CMF and was at the pre-exposure level during the post-exposure period. In two rats, pretreated with the vehicle on Day 1, CMF increased a hippocampal beta(2) level. The effect was about half the amplitude and shorter in duration than that after agroclavine, with no effects in the post-exposure period. However, during the 2nd exposure to CMF, started 30 min later, cortical and hippocampal beta activity was significantly greater than that during the 1st. On Day 10, the 1st exposure was completely ineffective, while during the 2nd, the hippocampal beta(2) activity was increased gradually. These results demonstrate that dopaminergic activation can potentiate CMF-evoked EEG effects in the hippocampal beta(2) frequency band.

Topics: Animals; Cerebral Cortex; Dopamine Agonists; Electroencephalography; Ergolines; Hippocampus; Magnetics; Male; Rats; Rats, Wistar

The grass parasites Claviceps purpurea and Claviceps fusiformis produce ergot alkaloids (EA) in planta and in submerged culture. Whereas EA synthesis (EAS) in C. purpurea proceeds via clavine intermediates to lysergic acid and the complex ergopeptines, C. fusiformis produces only agroclavine and elymoclavine. In C. purpurea the EAS gene (EAS) cluster includes dmaW (encoding the first pathway step), cloA (elymoclavine oxidation to lysergic acid), and the lpsA/lpsB genes (ergopeptine formation). We analyzed the corresponding C. fusiformis EAS cluster to investigate the evolutionary basis for chemotypic differences between the Claviceps species. Other than three peptide synthetase genes (lpsC and the tandem paralogues lpsA1 and lpsA2), homologues of all C. purpurea EAS genes were identified in C. fusiformis, including homologues of lpsB and cloA, which in C. purpurea encode enzymes for steps after clavine synthesis. Rearrangement of the cluster was evident around lpsB, which is truncated in C. fusiformis. This and several frameshift mutations render CflpsB a pseudogene (CflpsB(Psi)). No obvious inactivating mutation was identified in CfcloA. All C. fusiformis EAS genes, including CflpsB(Psi) and CfcloA, were expressed in culture. Cross-complementation analyses demonstrated that CfcloA and CflpsB(Psi) were expressed in C. purpurea but did not encode functional enzymes. In contrast, CpcloA catalyzed lysergic acid biosynthesis in C. fusiformis, indicating that C. fusiformis terminates its EAS pathway at elymoclavine because the cloA gene product is inactive. We propose that the C. fusiformis EAS cluster evolved from a more complete cluster by loss of some lps genes and by rearrangements and mutations inactivating lpsB and cloA.

Topics: Biosynthetic Pathways; Chromatography, Liquid; Chromatography, Thin Layer; Claviceps; Ergolines; Ergot Alkaloids; Evolution, Molecular; Gene Order; Genes, Fungal; Mass Spectrometry; Models, Biological; Molecular Structure; Multigene Family; Mutation; Species Specificity

This report presents results obtained from studies of the mnemotropic activity of the ergot alkaloid agroclavin. The effects of this substance were studied at concentrations of 1, 10, 25, 50, and 200 microg/kg on the learning and retention of a spatial habit in a Morris water maze. These doses of agroclavin had no effect on the process of formation of the spatial habit, but sharply disturbed retention of the habit. A long-lasting effect of memory trace disturbance was seen over a period of 48 h after dosage. On retraining, animals showed no delayed effects with agroclavin on learning a new spatial habit. The possible mechanisms for the effect of agroclavin on memory are discussed.

Topics: Animals; Dopamine Agonists; Dose-Response Relationship, Drug; Ergolines; Male; Maze Learning; Memory; Mice; Serial Learning; Space Perception

The effects of the dopamine receptor agonist agroclavin on cognitive processes associated with mechanisms of visual recognition and long-term and working (short-term) memory were studied in delayed visual differentiation and delayed spatial selection tasks in monkeys (rhesus macaques). Measurements made before and after p.o. pharmacological treatment with this agent were used to identify the p.o. dose (5 mg/kg) inducing a significant effect. The psychotropic effect of agroclavin, which induced cognitive dysfunction, was present in all the monkeys studied to one extent or another. Behavioral criteria were: the probability of correct solutions of the visual differentiation task, the probability of refusals to resolve the task, and the time taken for correct motor responses. Despite individual differences in these behavioral characteristics in monkeys, significant changes due to agroclavin were consistently evident in all animals. There was a reduction in the probability of correct solutions, due to worsening of the characteristics of short-term memory; most monkeys showed increases in the numbers of refusals to solve tasks and increases in the time for correct motor responses during these solutions. In fact, all monkeys showed no increases in the number of erroneous solutions in visual differeniation and spatial selection tasks without delays, though in most cases there were increases in the time taken for correct motor reactions and the number of refusals to solve tasks. Data were obtained indicating that the effect of agroclavin was not uniform with respect to different types of visual information. The possible structural-functional organization of processes underlying working memory is discussed on the basis of the conclusion that the behavioral characteristics studied here reflect different components of cognitive processes realized by structures with different functional properties and different locations.

Topics: Animals; Conditioning, Operant; Discrimination, Psychological; Dopamine Agonists; Ergolines; Macaca mulatta; Memory, Short-Term; Psychomotor Performance; Reaction Time; Visual Perception

Different culture media containing different effectors for production of ergot-alkaloids were prepared. The culture media were inoculated with equal mycelia of Claviceps purpurea and some were incubated for 22 days and some incubated for 45 days. The medium that contained a mixture of glucose, glycine and tryptophan showed highest yield of the alkaloids and reached the maximum period for production of agroclavine faster.

Topics: Antibiotics, Antineoplastic; Claviceps; Culture Media; Ergolines; Ergot Alkaloids

Neurochemical mechanisms of the effect of agroclavin on spatial memory in the Morris water maze in mice were studied by analyzing the effect of neurotransmitter receptor ligands on the amnestic effect of this alkaloid. D1-receptor agonist SKF-38393 and calcium channel blockers verapamil and nimodipine abolished the negative effect of agroclavin on spatial memory. The role of intracellular calcium in the mechanisms of amnestic effect of agroclavin is discussed.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Amnesia; Animals; Dopamine Agonists; Ergolines; Maze Learning; Mice; Receptors, Dopamine D1

Dimers of agroclavine (1) and terguride (2), as well as a series of terguride oligomers, for example trimers (5, 6), tetramer (7), hexamer (8) and functionalized tergurides for further complex clustering were synthesized. Terguride oligomers were screened for their direct cellular toxicity on lymphoma cell lines in vitro and for their immunomodulating activities, represented by the natural killer (NK) cell-mediated cytotoxicity, as the most sensitive screening marker during immune responses. Dimers linked via aromatic spacer showed a high toxicity (1 microM) to lymphoma cells, which was not detected in other derivatives. In vitro and ex vivo experiments performed on mouse spleen lymphocytes in the presence of terguride oligomers demonstrated an immunosuppressive effect of dimers with aromatic spacer (4c-d) and NK cell stimulatory effect of terguride hexamer (8) and trimer with aliphatic spacer (5c). There is a considerable evidence that indolic part of molecule contributes to immunosuppressive action of terguride, which is potentiated in dimers carrying aromatic linker. This effect can be reversed by higher oligomerization of the respective alkaloids.

Topics: Adjuvants, Immunologic; Animals; Carbamates; Dimerization; Drug Evaluation, Preclinical; Ergolines; Female; Humans; Immunosuppressive Agents; Killer Cells, Natural; Lisuride; Mice; Mice, Inbred BALB C; Spleen; Structure-Activity Relationship; Tumor Cells, Cultured

Agroclavine is a natural, clavine type of ergot alkaloid with D1 dopamine and a-adrenoceptor agonistic properties. We showed previously that in vitro agroclavine enhances natural killer (NK) cell activity, increases interleukin-2 and interferon-gamma production and prolongs the survival time of tumor-bearing mice. The aim of this study was 1) to test the effect of agroclavine on NK activity in vivo, and 2) to assess the potential toxicity of high doses of agroclavine on cardiac and liver functions using creatine kinase MB (CKMB) and alanine aminotransferase (ALT) as biochemical markers in normal and stressed animals. The effect of stress was studied because we examined promising anticancer properties of agroclavine and malignant diseases are supposed to be a potent stressful event for patients. In our experiments 3-month-old male rats of the Wistar-Kyoto strain were used. Agroclavine was injected intraperitoneally (0.5 mg/kg or 0.05 mg/kg) 30 min before stress (four hours' restraint and immersion in 23 degrees C water). The animals were killed 30 min after stress, blood was collected and the spleen was removed. Non-stressed animals treated with agroclavine were killed 5 h after the drug administration. The results confirmed our previous in vitro results and showed that also in vivo agroclavine increases NK cell activity under non-stress conditions. Agroclavine only slightly increased CKMB and had no influence on ALT in non-stressed animals. These promising results are limited by the fact that agroclavine (0.5 mg/kg) diminished NK cell activity and significantly increased ALT and CKMB under stress conditions.

Topics: Alanine Transaminase; Animals; Antibiotics, Antineoplastic; Chemical and Drug Induced Liver Injury; Creatine Kinase; Ergolines; Heart Diseases; Immersion; Injections, Intraperitoneal; Killer Cells, Natural; Liver Diseases; Male; Rats; Rats, Inbred WKY; Restraint, Physical; Stress, Physiological

Prior to and after pharmacological effect of Agroclavine (a dopamine receptors' agonist) following its per os administration, its optimal dose (5 mg/kg) for cognitive processes was established in monkeys. Psychotropic effect of Agroclavine resulting in cognitive dysfunction manifested itself to a greater of lesser extent in all the experimental animals. Correctness of visual differentiation, probability of refusal to solve a task, time of a correct motor response were assumed as the behavioural criteria. In fact, all the animals revealed absence of any increase in the number of wrong solutions in visual differentiation or spatial choice, although the time of correct morot response and the number of refusal to solve the task mostly increased. The Agroclavine effect was found to be rather variable in respect to different types of visual information. A possible structural-functional organisation of the working memory processes is discussed.

Topics: Animals; Cognition; Dopamine Agonists; Ergolines; Macaca mulatta; Memory, Short-Term; Motivation; Psychomotor Performance; Reaction Time; Visual Perception

Ergot alkaloids (EAs), products of Claviceps spp., are widely used in various fields of clinical medicine (neurology, psychiatry, endocrinology). In the present work we studied the neuroimmunomodulative effect of EAs on activation of NK cells and their signalling pathways. Furthermore, the killing capability of rat NK cells in vitro was examined in the presence of glycosidic derivatives of elymoclavine, agroclavine, and liposome-encapsulated EAs. The engagement of appropriate NK cell membrane receptors by EAs cause an indirect enhancement of adenylyl cyclase system through inhibition of G-protein al,2-subunit (up to 50 % of control values). All of the tested EAs enhanced the rat NK cell-mediated cytotoxic activity in vitro, particularly against target cells of astrocyte origin (C-6 glioma). The present results argue for a possible EA immunomodulatory role of cell-mediated immunity in tumour regression processes.

Topics: Animals; Cell Division; Cells, Cultured; Claviceps; Cytotoxicity, Immunologic; Drug Delivery Systems; Ergolines; Ergot Alkaloids; Humans; Killer Cells, Natural; Liposomes; Mice; Monocytes; Neuroimmunomodulation; Oligosaccharides; Rats; Signal Transduction; Tumor Cells, Cultured

Topics: Cell Division; Cells, Cultured; Coculture Techniques; Cytotoxicity, Immunologic; DNA Replication; Drug Synergism; Ergolines; Ergot Alkaloids; Humans; Interferon-gamma; Interleukin-2; Lymphocyte Activation; Lymphocytes; Lymphokines; Mitogens; Phytohemagglutinins; Recombinant Proteins

Effects of festuclavine derivatives on nucleoside uptake by human lymphoid leukemia Molt 4B cells and on incorporation into TCA-insoluble materials in the cells were examined. The uptake and incorporation of uridine or thymidine were suppressed by festuclavine (EN01), 13-bromo-1-cyclopropylmethyl-festuclavine (EN02), 1-(4-chloro-benzenesulfonyl)festuclavine (EN03) and 1-cyclopentyl festuclavine (EN04) at 10-50 microM. Among these compounds, EN02 was most effective and at 50 microM it completely suppressed cellular uptake of the nucleosides and their incorporation into TCA-insoluble materials inhibiting the cellular proliferation. EN03 and EN04 moderately inhibited the transport and incorporation of the nucleosides in dose-dependent manners, while the mother compound EN01 had the least inhibitory effect. These findings indicated that alkylation at the indole nitrogen in combination with bromination at C-13 of the festuclavine molecule strengthened its inhibitory action on nucleoside uptake to a remarkable extent. The inhibition curves of nucleoside incorporation into TCA-insoluble materials showed quite similar dose-dependence to those of the inhibition curves for cellular nucleoside transport. These results suggest that the inhibitions of DNA and RNA syntheses by the festuclavine derivatives are due to the depressed transport of nucleosides into the leukemia cells.

Topics: Antibiotics, Antineoplastic; Cell Division; Chemical Phenomena; Chemistry; Depression, Chemical; DNA, Neoplasm; Dose-Response Relationship, Drug; Ergolines; Humans; Leukemia, Lymphoid; Nucleosides; RNA, Neoplasm; Thymidine; Tumor Cells, Cultured; Uridine

We determined the vasoconstrictive effects of selected ergot alkaloids, and a sample containing loline and its derivative alkaloids, on the isolated dorsal pedal vein of cattle, as a model system to study one of the toxic effects that result from cattle ingesting fescue forage infected with the endophytic fungus Acremonium coenophilalum. The ergot compounds ergotamine, ergosine, and agroclavine constricted this peripheral vein of cattle, but much less so than did the alpha-adrenergic agonist norepinephrine, which supports the ergots acting as partial agonists for these receptors. However, the sample of loline and loline-derivative alkaloids did not affect the dorsal pedal vein when given at concentrations similar to those of the ergot compounds. Loline and loline-derivative alkaloid sample at high concentrations partially inhibited norepinephrine-elicited vascular contraction, an effect that appeared to be unrelated to alpha-adrenoceptor activity. Thus, in the dorsal pedal vein model in cattle, the ergopeptide alkaloids were more venoconstrictive than were loline and its derivative alkaloids.

Topics: Acremonium; Alkaloids; Animals; Cattle; Dose-Response Relationship, Drug; Ergolines; Ergot Alkaloids; Ergotamine; Ergotamines; Hindlimb; Norepinephrine; Poaceae; Vasoconstriction; Veins

Agroclavine, an alkaloid produced by some species of fungi and dicotyledon plants, and its 1-alkylated derivatives are potentially useful as antineoplastic drugs, since they exert potent and selective cytostatic effects. In the present study, we have investigated agroclavine and its 1-propyl and 1-pentyl derivatives for mutagenicity. The genetic end point studied was the reversion of strains of Salmonella typhimurium (TA 100, TA 98, TA 1537) and Escherichia coli (WP2 uvrA), auxotrophic for histidine and tryptophan, respectively. The compounds were tested directly and in the presence of a mammalian xenobiotic-metabolizing system. In the direct test, agroclavine and the two alkylated derivatives examined exhibited substantial bacteriotoxicity but no mutagenicity. Addition of NADPH-fortified postmitochondrial supernatant fraction of rat liver homogenate led to a clear-cut decrease in bacteriotoxicity and to the formation of mutagenic products. Each compound was effective in all three strains of S. typhimurium used. In E. coli only spurious effects were seen. 1-Pentylagroclavine, the most hydrophobic compound in the series, was the strongest mutagen. Agroclavine, the least hydrophobic compound, was the weakest. The mutagenic potencies and efficacies of all these test compounds were much weaker than those of the positive controls, which were known mutagens and carcinogens. Moreover, the differential effect of metabolism by liver enzymes demonstrates that the toxicity and mutagenicity of agroclavine and its derivatives are caused by different chemical species. Hence, it may be possible to develop derivatives that are cytotoxic but not mutagenic.

Topics: Animals; Biotransformation; Carcinogens; Ergolines; Escherichia coli; Male; Microsomes, Liver; Mutagenicity Tests; Mutagens; Mutation; Rats; Rats, Inbred Strains; Salmonella typhimurium

The cytostatic potential of twenty antibiotic agroclavines has been examined in the L5178y mouse lymphoma cell system. Twelve of these compounds are described for the first time. It is shown that the substituent at N-1 of agroclavine is very important whereas the substituent at N-6 is of less influence if it is not hydrogen. Incorporation studies in the presence of 1-propylagroclavine suggest that DNA synthesis in the lymphoma cells is inhibited. The effect on the corresponding [3H]thymidine incorporation in murine spleen lymphocytes is comparably low. Neither a significant change of mRNA efflux nor of DNA polymerase alpha and beta activities was caused. The mechanism of action seems to be a fundamentally new one for ergoline compounds as interactions with alpha-adrenoceptors, dopamine and 5-hydroxytryptamine receptors are not involved.

Topics: Animals; Antibiotics, Antineoplastic; DNA-Directed DNA Polymerase; DNA, Neoplasm; Ergolines; Leukemia L1210; Lymphocyte Activation; Male; Mice; Neoplasm Proteins; Receptors, Dopamine; Receptors, Serotonin; RNA, Messenger; RNA, Neoplasm; Structure-Activity Relationship

In submerged Claviceps cultures the activity of hydroxymethylglutaryl-CoA reductase preceded the increase of alkaloid production and of sterol content. During the first alkaloid phase, cell mevalonate was involved in the biosynthesis of both alkaloids and steroids. In the second production phase, it was predominantly used for alkaloid synthesis. Hydroxymethylglutaryl-CoA reductase appears to be a suitable target for physiological manipulation to increase clavine alkaloid yields.

Topics: Claviceps; Ergolines; Ergot Alkaloids; Hydroxymethylglutaryl CoA Reductases; Sterols

Agroclavine, given to actively-growing sclerotial tissue of a strain of Claviceps purpurea which can not normally elaborate ergot alkaloids, was transformed by this tissue into lysergic acid amide with overall efficiency of approximately 40%. By contrast, festuclavine (8,9-dihydro-agroclavine) was not transformed, indicating specificity in the mechanism of lysergyl biosynthesis.

Topics: Claviceps; Ergolines; Lysergic Acid Diethylamide; Structure-Activity Relationship

L-Tryptophan did not exert any influence on peptide alkaloid formation in an ergotamine and in an ergosine-accumulating C. purpurea strain. A different picture was observed in a series of related C. purpurea strains. Tryptophan showed a slight stimulatory effect on the ergotoxine producer Pepty 695/S. A blocked mutant of it, designated as Pepty 695/ch which was able to accumulate secoclavines gave similar results. In a high-yielding elymoclavine strain Pepty 695/e, the progeny of the former one, tryptophan up to a concentration of 25 mM stimulated remarkably clavine biosynthesis. Furthermore, tryptophan could overcome the block of synthesis by inorganic phosphate. Increased specific activities of chanoclavine cyclase but not DMAT synthetase were observed in cultures of strain Pepty 695/e supplemented with tryptophan. 5-Methyltryptophan and bioisosteres of tryptophan were ineffective in alkaloid stimulation. These results are compared with those obtained with the grass ergot strain SD 58 and discussed with the relation to other induction phenomena.

Topics: Chemical Phenomena; Chemistry; Claviceps; Culture Media; Ergolines; Ergot Alkaloids; Mutation; Phosphates; Stereoisomerism; Tryptophan

Topics: Cardiovascular Agents; Colorimetry; Countercurrent Distribution; Ergolines; Ergot Alkaloids; Oxytocics

Topics: Cardiovascular Agents; Countercurrent Distribution; Ergolines; Ergot Alkaloids; Humans; Oxytocics