Ergot cluster-encoded catalase is required for synthesis of chanoclavine-I in Aspergillus fumigatus.

Genes required for ergot alkaloid biosynthesis are clustered in the genomes of several fungi. Several conserved ergot cluster genes have been hypothesized, and in some cases demonstrated, to encode early steps of the pathway shared among fungi that ultimately make different ergot alkaloid end products. The deduced amino acid sequence of one of these conserved genes (easC) indicates a catalase as the product, but a role for a catalase in the ergot alkaloid pathway has not been established. We disrupted easC of Aspergillus fumigatus by homologous recombination with a truncated copy of that gene. The resulting mutant (ΔeasC) failed to produce the ergot alkaloids typically observed in A. fumigatus, including chanoclavine-I, festuclavine, and fumigaclavines B, A, and C. The ΔeasC mutant instead accumulated N-methyl-4-dimethylallyltryptophan (N-Me-DMAT), an intermediate recently shown to accumulate in Claviceps purpurea strains mutated at ccsA (called easE in A. fumigatus) (Lorenz et al. Appl Environ Microbiol 76:1822-1830, 2010). A ΔeasE disruption mutant of A. fumigatus also failed to accumulate chanoclavine-I and downstream ergot alkaloids and, instead, accumulated N-Me-DMAT. Feeding chanoclavine-I to the ΔeasC mutant restored ergot alkaloid production. Complementation of either ΔeasC or ΔeasE mutants with the respective wild-type allele also restored ergot alkaloid production. The easC gene was expressed in Escherichia coli, and the protein product displayed in vitro catalase activity with H(2)O(2) but did not act, in isolation, on N-Me-DMAT as substrate. The data indicate that the products of both easC (catalase) and easE (FAD-dependent oxidoreductase) are required for conversion of N-Me-DMAT to chanoclavine-I.

Apical sensory neurones mediate developmental retardation induced by conspecific environmental stimuli in freshwater pulmonate snails.

Freshwater pond snails Helisoma trivolvis and Lymnaea stagnalis undergo larval development and metamorphosis inside egg capsules. We report that their development is permanently under slight tonic inhibitory influence of the anterior sensory monoaminergic neurones, which are the remnants of the apical sensory organ. Conspecific juvenile snails, when reared under conditions of starvation and crowding, release chemical signals that are detected by these neurones in encapsulated larvae and reversibly suppress larval development, thus providing a link between environmental signals and developmental regulation. Induced retardation starts from the trochophore stage and results in up to twofold prolongation of the larval lifespan. Upon stimulation with the signal, the neurones increase synthesis and release of monoamines [serotonin (5-HT) in Helisoma and dopamine in Lymnaea] that inhibit larval development acting via ergometrine-sensitive internal receptors. Thus, the novel regulatory mechanism in larval development of molluscs is suggested and compared with the phenomenon of dauer larvae formation in the nematode Caenorhabditis elegans.

[Isolation of the ergot (Claviceps purpurea (Fr.) Tul., strain VKM-F-366D), producing the lactamic alkaloid ergocornam]. / Vydelenie shtamma sporyn'i (Claviceps purpurea (Fr.) Tul., BKM-F-3662D), produtsiruiushchego alkaloid laktamnogo riada--épgokornam.

A new ergot strain VKM-F-3662D producing lactamic alkaloid ergocornam with concomitant alkaloids valinamide and ergometrine was isolated during selective works with sclerotium MS-462, which was obtained from ergocryptine ergot strain VKM-F-2642D. The structure of these alkaloids was determined by 1H and 13C NMR.

Hydrolysis of lysergamide to lysergic acid by Rhodococcus equi A4.

From a mixture of lysergamide and its epimer isolysergamide, Rhodococcus equi A4 containing amidase preferentially hydrolyzed lysergamide into lysergic acid.

Alkaloid binding and activation of D2 dopamine receptors in cell culture.

Ergot and pyrrolizidine alkaloids, either extracted from endophyte-infected tall fescue, synthesized, or purchased commercially, were evaluated in cultured cells to estimate their binding to the D2 dopamine receptor and subsequent effects on cyclic AMP production in GH4ZR7 cells, transfected with a rat D2 dopamine receptor. Ergopeptide alkaloid (alpha-ergocryptine, bromocryptine, ergotamine tartrate, and ergovaline) inhibition of the binding of the D2-specific radioligand, [3H]YM-09151-2, exhibited inhibition constants (K(I)) in the nanomolar range, whereas dopamine was less potent (micromolar). The lysergic acid amides (ergine and ergonovine) were 1/100th as potent as the ergopeptide alkaloids. Ergovaline and ergotamine tartrate were equally effective in inhibiting vasoactive intestinal peptide (VIP)-stimulated cyclic AMP production, with consistent nanomolar effective concentration (EC50) values. The remaining ergopeptide alkaloids (alpha-ergocryptine and bromocryptine), lysergic acid amides (ergonovine and ergine), and dopamine were 1/100th as potent. Two representative pyrrolizidines, N-formylloline and N-acetylloline, exhibited no binding activity at the D2 dopamine receptor or effects on the cyclic AMP system within the concentration ranges of nanomolar to millimolar. Our results indicate that the commercially available ergot alkaloids ergotamine tartrate and ergonovine may be used interchangeably in the D2 dopamine receptor system to simulate the effects of extracted ergovaline and ergine and to examine responses in receptor binding and the inhibition of cyclic AMP.

Development and validation of a nonisotopic immunoassay for the detection of LSD in human urine.

A microplate enzyme immunoassay (EIA) for the detection of lysergic acid diethylamide (LSD) in human urine was developed. The assay kit is designed around an LSD derivative coated on the wall of microplate wells with preservatives and stabilizers. Sample and rabbit anti-LSD are added to the microplate well. The immobilized LSD and LSD present in specimens compete for the opportunity to bind to the anti-LSD antibodies. An anti-rabbit antibody labeled with horseradish peroxidase is used to provide the assay signal, which is inversely proportional to the concentration of LSD in the sample. The assay requires a 25-microL urine sample and three consecutive incubation periods of 60, 30, and 30 min at room temperature. The assay was tested with a variety of drugs, including ergot alkaloids spiked into drug-free urine at up to 100,000 ng/mL without cross-reaction. Nor-LSD was shown to cross-react between 16% and 28%, depending on its concentration. Of the other compounds tested, only ergonovine demonstrated slight cross-reactivity at approximately 0.0008%. The assay is designed to be used with a qualitative cutoff of 0.5 ng/mL. Precision testing at 0.5 ng/mL gave a coefficient of variation (CV) of 6% based on 20 replicates. The CV at 0.375 ng/mL (cutoff, -25%) was 5.2% and at 0.625 ng/mL was 6.6%. Precision at other concentrations within the range of the calibration curve gave similar results both intra- and interassay. Clinical performance of the assay was compared with that of a commercial radioimmunoassay (RIA). Comparable performance was observed with both methods, each screening a total of 458 samples as negative and 17 samples as positive relative to a 0.5 ng/mL cutoff. The EIA found an additional three positive samples that were negative by RIA. The EIA is suitable for the screening of urine samples for the presence of LSD. Preliminary indications are that the assay is also suitable for use with whole blood specimens. The assay can be performed manually or be fully automated and without the need for radioactivity; it can be used in any laboratory.

Ergometrine and methylergometrine tablets are not stable under simulated tropical conditions.

OBJECTIVES: This study is part of a programme on reduction of postpartum haemorrhage (PPH). Ergometrine and methylergometrine have a favourable effect on both blood loss and maternal morbidity and mortality and oral preparations were regarded as a possible treatment for use in tropical countries. The stability of oral preparations of the two ergometrine compounds under tropical conditions was unknown and was therefore examined in this study. STUDY METHODS: The 'experimental shelf lives' of ergometrine and methylergometrine tablets were examined by exposing the tablets to seven artificially controlled conditions. Samples were analysed by high performance liquid chromatography at nine different sampling times over a period of 1 year to determine the content of ergometrine and methylergometrine. RESULTS: Under refrigeration (test I), less than 90% of the stated amount of active ingredient was found in the tablets after 14 weeks in the case of ergometrine and 21 weeks in the case of methylergometrine. When stored in the dark at 40 degrees C and 75% relative humidity (test VI), the tablets fall outside accepted specification (= 90-110% of state amount of active ingredient) within 3 weeks in the case of ergometrine and 21 weeks in the case of coated methylergometrine tablets. The stability of uncoated ergometrine tablets was far less than that of coated methylergometrine tablets. Instability worsened under extreme humid conditions (test IV and VI), and hot conditions (test V), for both ergometrine and methylergometrine. From week 31 onwards the coating did not seem to protect the compound anymore, irrespective of the condition of exposure. CONCLUSIONS: Tropical conditions make the tablets unstable with humidity as the main adverse factor. The sugar-coated methylergometrine tablets are more stable under humid/hot conditions than the non-coated ergometrine tablets. Under all simulated conditions both oral ergometrine and methylergometrine tablets are unstable.

N(1)-substituted ergolines and tryptamines show species differences for the agonist-labeled 5-HT2 receptor.

Previous studies indicated that selected ergolines and tryptamines showed species differences for affinity to the antagonist-labeled 5-HT2 receptor. The present study examined these same compounds for affinity at the agonist-labeled 5-HT2 receptor in rat and squirrel monkey cortical homogenates using [125I]DOI ([125I]1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane). As seen with the antagonist-labeled 5-HT2 receptor, N(1) alkyl substitution of either the ergolines or tryptamines resulted in a slight increase or no effect on their affinity for the agonist-labeled rat 5-HT2 receptor. In contrast, these same N(1) substitutions resulted in significant decreases in affinity for the agonist-labeled monkey 5-HT2 receptor. It was also noted that N(1)-unsubstituted ergolines and tryptamines (such as ergonovine, LY86057, LY193525 and 5-methoxytryptamine) tended to have higher affinity for the monkey versus the rat agonist-labeled receptor. However, the N(1) alkyl-substituted ergolines and tryptamines (such as mesulergine, LY53857, amesergide, N(1)-isopropyltryptamine and N(1)-isopropyl-5-methoxytryptamine) showed significantly lower affinity for the monkey versus the rat 5-HT2 receptor. These data suggest that, at least in relation to the N(1) position, ergolines and tryptamines bind in a similar orientation. These results are also discussed in terms of what amino acid differences between species may account for this structure-activity relationship.

Ergometrine and 5-hydroxytryptamine binding sites in rat brain and myometrium.

Functional studies suggest that ergometrine is a partial agonist involving 5-hydroxytryptamine (5-HT) receptors in rat uterus. Ergometrine displaced [3H]5-HT from specific binding sites in rat brain, but did not displace [3H]5-HT at functionally important concentrations in rat myometrium. These binding studies indicate that the agonist and antagonist actions of ergometrine in rat uterus arise from its initial interaction with binding sites other than those for 5-HT.