Large-Scale Isolation of Lolitrem B and Structure Determination of Lolitrem E

Miles, Christopher O.; Munday, Sarah C.; Wilkins, Alistair L.; Ede, Richard M.; Towers, N. R.

doi:10.1021/jf00043a018

Cited by 56 publications

(76 citation statements)

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“…Lolitrem B was extracted from ryegrass seed infected with Neotyphodium lolii and purified by chemical methods as described previously (Miles et al, 1994). The resulting crystalline solid was shown to be at least 97% pure by NMR spectroscopy.…”

Section: Methodsmentioning

confidence: 99%

“…The intensity of tremor induced by the toxins was measured using a previously described mouse bioassay method (Gallagher and Hawkes, 1986). This bioassay has been used extensively in our laboratories during structure-activity studies of tremorgenic toxins (Miles et al, 1992(Miles et al, , 1994Munday-Finch et al, 1997). Toxins were administered by intraperitoneal injection as a solution in 9:1 (v/v) dimethyl sulfoxide (DMSO)-water (50 l).…”

Section: Methodsmentioning

confidence: 99%

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The Molecular Mechanism of “Ryegrass Staggers,” a Neurological Disorder of K⁺ Channels

Imlach

Finch

Dunlop

et al. 2008

J Pharmacol Exp Ther

103

115

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"Ryegrass staggers" is a neurological condition of unknown mechanism that impairs motor function in livestock. It is caused by infection of perennial ryegrass pastures by an endophytic fungus that produces neurotoxins, predominantly the indolediterpenoid compound lolitrem B. Animals grazing on such pastures develop uncontrollable tremors and become uncoordinated in their movement. Lolitrem B and the structurally related tremor inducer paxilline both act as potent large conductance calcium-activated potassium (BK) channel inhibitors. Using patch clamping, we show that their different apparent affinities correlate with their toxicity in vivo. To investigate whether the motor function deficits produced by lolitrem B and paxilline are due to inhibition of BK ion channels, their ability to induce tremor and ataxia in mice deficient in this ion channel (Kcnma1 Ϫ/Ϫ ) was examined. Our results show that mice lacking Kcnma1 are unaffected by these neurotoxins. Furthermore, doses of these substances known to be lethal to wild-type mice had no effect on Kcnma1 Ϫ/Ϫ mice. These studies reveal the BK channel as the molecular target for the major components of the motor impairments induced by ryegrass neurotoxins. Unexpectedly, when the response to lolitrem B was examined in mice lacking the ␤4 BK channel accessory subunit (Kcnmb4 Ϫ/Ϫ ), only low-level ataxia was observed. Our study therefore reveals a new role for the accessory BK ␤4 subunit in motor control. The ␤4 subunit could be considered as a potential target for treatment of ataxic conditions in animals and in humans.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Molecular Mechanism of “Ryegrass Staggers,” a Neurological Disorder of K⁺ Channels

Imlach

Finch

Dunlop

et al. 2008

J Pharmacol Exp Ther

103

115

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“…Quantification was achieved by comparison with a pure standard (Miles et al 1994). The limit of quantitation for this technique is 0.1 mg/kg.…”

Section: Endophyte Alkaloid Analysismentioning

confidence: 99%

The occurrence of ryegrass staggers and heat stress in sheep grazing ryegrass-endophyte associations with diverse alkaloid profiles

Fletcher

Finch

Sutherland

et al. 2017

New Zealand Veterinary Journal

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AIMS: To compare ryegrass pastures infected with endophytes producing diverse alkaloids for their ability to cause ryegrass staggers in grazing lambs; to compare respiration rates and rectal temperatures of these lambs after exposure to heat stress, and to compare liveweight gains during the study period.METHODS: Ryegrass pastures of cultivar Trojan infected with NEA endophytes, branded NEA2 (T-NEA2), endophyte-free Trojan (T-NIL), Samson infected with standard endophyte (S-STD), Samson infected with AR37 endophyte (S-AR37) and endophyte-free Samson (S-NIL), were grazed by lambs (n=30 per cultivar) for up to 48 days in February and March of 2012 and 2013. Pasture samples were analysed for alkaloid concentrations and lambs were scored for ryegrass staggers at intervals during the study period. Liveweight was recorded at the start (Day 0) and end of the study, and rectal temperatures and respiratory rates were measured in lambs exposed to heat stress on Days 23 and 26, in 2012 and 2013, respectively. RESULTS: Concentrations of alkaloids were lower in 2012 than 2013, associated with warmer and drier conditions in 2013, and the prevalence of ryegrass staggers was low in 2012. In 2013, concentrations of ergovaline were similar in T-NEA2 and S-STD, but concentrations of lolitrem B were lower in T-NEA2 than S-STD. S-AR37 produced epoxy-janthitrems but no lolitrem B or ergovaline. In 2013, by Day 20, 9/30 (30%) sheep grazing S-STD had severe staggers (score ≥4), and by Day 47 all sheep had been removed from this cultivar due to severe staggers. By Day 47, 18/30 (60%), 4/30 (13%) and 0/ 30 (0%) sheep grazing S-AR37, T-NEA2 and T-NIL pastures, respectively, had severe staggers. There were no differences in mean daily weight gain of lambs between cultivars in either year. In both years, mean rectal temperature and respiration rate following exposure to heat stress were highest in sheep grazing S-STD and T-NEA2, and lowest in sheep grazing T-NIL. CONCLUSIONS: In lambs grazing different ryegrass pastures infected with endophytes, ryegrass staggers was most severe on S-STD, less severe on S-AR37 and least on T-NEA2. When under heat stress, lambs grazing ergovaline-producing S-STD and T-NEA2 pastures had increased respiration rates and rectal temperatures compared with lambs grazing T-NIL.CLINICAL RELEVANCE: If ambient temperatures are suitable, NEA2-branded endophytes have the potential to express concentrations of ergovaline sufficient to induce heat stress in grazing sheep.

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“…The absence of lolitrems in many epichloë endophyte-grass associations does not appear to be due to a grass host effect but rather to the inability of the endophyte to produce the compound (5, 63). Until recently, genetic analysis of the genes required for the production of lolitrem B was not available, so a large effort was put into the chemical analysis of endophyte-infected grasses that resulted in the identification of indole-diterpene compounds, such as lolitriol, lolicines A and B, lolilline and terpendole M, predicted to be precursors or by-products of lolitrem B biosynthesis (5,19,37,38,40,(46)(47)(48)(49). These analyses showed that the ability to produce the alkaloids was dependent on the presence of the endophyte within the plant.…”

mentioning

confidence: 99%

Indole-Diterpene Biosynthetic Capability ofEpichloëEndophytes as Predicted byltmGene Analysis

Young

Tapper

May

et al. 2009

Appl Environ Microbiol

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Bioprotective alkaloids produced by Epichloë and closely related asexual Neotyphodium fungal endophytes protect their grass hosts from insect and mammalian herbivory. One class of these compounds, known for antimammalian toxicity, is the indole-diterpenes. The LTM locus of Neotyphodium lolii (Lp19) and Epichloë festuce (Fl1), required for the biosynthesis of the indole-diterpene lolitrem, consists of 10 ltm genes. We have used PCR and Southern analysis to screen a broad taxonomic range of 44 endophyte isolates to determine why indole-diterpenes are present in so few endophyte-grass associations in comparison to that of the other bioprotective alkaloids, which are more widespread among the endophtyes. All 10 ltm genes were present in only three epichloë endophytes. A predominance of the asexual Neotyphodium spp. examined contained 8 of the 10 ltm genes, with only one N. lolii containing the entire LTM locus and the ability to produce lolitrems. Liquid chromatography-tandem mass spectrometry profiles of indole-diterpenes from a subset of endophyte-infected perennial ryegrass showed that endophytes that contained functional genes present in ltm clusters 1 and 2 were capable of producing simple indole-diterpenes such as paspaline, 13-desoxypaxilline, and terpendoles, compounds predicted to be precursors of lolitrem B. Analysis of toxin biosynthesis genes by PCR now enables a diagnostic method to screen endophytes for both beneficial and detrimental alkaloids and can be used as a resource for screening isolates required for forage improvement.Epichloë endophytes systemically colonize cool-season grasses, and comprise sexual Epichloë spp. and their asexual derivatives, the Neoptyphodium spp. Collectively, they produce a range of bioprotective alkaloids, including the ergot alkaloids, pyrrolopyrazine (peramine), aminopyrrolizidines (lolines), and indole-diterpenes (including lolitrems) (5, 63). Most Neotyphodium spp. are of hybrid origin, with two or three Epichloë or Neotyphodium ancestors (11,44,59,66). The alkaloids produced by epichloë endophytes enhance the competitive ability of endophyte-infected grasses by protecting the grass host from insect and mammalian herbivory. Alkaloids such as peramine and the lolines are known for their antiinsect properties (5, 55, 63), while ergovaline and the lolitrems are detrimental to grazing livestock, causing fescue toxicosis and ryegrass staggers, respectively, in pastoral ecosystems (17, 18, 31, 47, 52). Many epichloë endophytes are able to produce multiple classes of alkaloids but, to date, no plant-endophyte combination has been identified that produces lolitrems, lolines, peramine, and ergovaline (5, 10).A number of naturally occurring endophytes have been identified that do not produce the mammalian toxins, ergovaline and lolitrem B, but retain beneficial agronomic properties (26, 27), such as tolerance to abiotic stress (32). Artificially inoculated associations of these endophytes with elite tall fescue (4) and perennial ryegrass cultivars (16) have been commercially e...

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Large-Scale Isolation of Lolitrem B and Structure Determination of Lolitrem E

Cited by 56 publications

References 6 publications

The Molecular Mechanism of “Ryegrass Staggers,” a Neurological Disorder of K⁺ Channels

The Molecular Mechanism of “Ryegrass Staggers,” a Neurological Disorder of K⁺ Channels

The occurrence of ryegrass staggers and heat stress in sheep grazing ryegrass-endophyte associations with diverse alkaloid profiles

Indole-Diterpene Biosynthetic Capability ofEpichloëEndophytes as Predicted byltmGene Analysis

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Large-Scale Isolation of Lolitrem B and Structure Determination of Lolitrem E

Cited by 56 publications

References 6 publications

The Molecular Mechanism of “Ryegrass Staggers,” a Neurological Disorder of K+ Channels

The Molecular Mechanism of “Ryegrass Staggers,” a Neurological Disorder of K+ Channels

The occurrence of ryegrass staggers and heat stress in sheep grazing ryegrass-endophyte associations with diverse alkaloid profiles

Indole-Diterpene Biosynthetic Capability ofEpichloëEndophytes as Predicted byltmGene Analysis

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The Molecular Mechanism of “Ryegrass Staggers,” a Neurological Disorder of K⁺ Channels

The Molecular Mechanism of “Ryegrass Staggers,” a Neurological Disorder of K⁺ Channels