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Publikation

Bochnia, M.; Sander, J.; Ziegler, J.; Terhardt, M.; Sander, S.; Janzen, N.; Cavalleri, J.-M. V.; Zuraw, A.; Wensch-Dorendorf, M.; Zeyner, A.; Detection of MCPG metabolites in horses with atypical myopathy PLOS ONE 14, e0211698, (2019) DOI: 10.1371/journal.pone.0211698

Atypical myopathy (AM) in horses is caused by ingestion of seeds of the Acer species (Sapindaceae family). Methylenecyclopropylacetyl-CoA (MCPA-CoA), derived from hypoglycin A (HGA), is currently the only active toxin in Acer pseudoplatanus or Acer negundo seeds related to AM outbreaks. However, seeds or arils of various Sapindaceae (e.g., ackee, lychee, mamoncillo, longan fruit) also contain methylenecyclopropylglycine (MCPG), which is a structural analogue of HGA that can cause hypoglycaemic encephalopathy in humans. The active poison formed from MCPG is methylenecyclopropylformyl-CoA (MCPF-CoA). MCPF-CoA and MCPA-CoA strongly inhibit enzymes that participate in β-oxidation and energy production from fat. The aim of our study was to investigate if MCPG is involved in Acer seed poisoning in horses. MCPG, as well as glycine and carnitine conjugates (MCPF-glycine, MCPF-carnitine), were quantified using high-performance liquid chromatography-tandem mass spectrometry of serum and urine from horses that had ingested Acer pseudoplatanus seeds and developed typical AM symptoms. The results were compared to those of healthy control horses. For comparison, HGA and its glycine and carnitine derivatives were also measured. Additionally, to assess the degree of enzyme inhibition of β-oxidation, several acyl glycines and acyl carnitines were included in the analysis. In addition to HGA and the specific toxic metabolites (MCPA-carnitine and MCPA-glycine), MCPG, MCPF-glycine and MCPF-carnitine were detected in the serum and urine of affected horses. Strong inhibition of β-oxidation was demonstrated by elevated concentrations of all acyl glycines and carnitines, but the highest correlations were observed between MCPF-carnitine and isobutyryl-carnitine (r = 0.93) as well as between MCPA- (and MCPF-) glycine and valeryl-glycine with r = 0.96 (and r = 0.87). As shown here, for biochemical analysis of atypical myopathy of horses, it is necessary to take MCPG and the corresponding metabolites into consideration.
Publikation

Bochnia, M.; Scheidemann, W.; Ziegler, J.; Sander, J.; Vollstedt, S.; Glatter, M.; Janzen, N.; Terhardt, M.; Zeyner, A.; Predictive value of hypoglycin A and methylencyclopropylacetic acid conjugates in a horse with atypical myopathy in comparison to its cograzing partners Equine Vet. Educ. 30, 24-28, (2018) DOI: 10.1111/eve.12596

Hypoglycin A (HGA) was detected in blood and urine of a horse suffering from atypical myopathy (AM; Day 2, serum, 8290 μg/l; urine: Day 1, 574, Day 2, 742 μg/l) and in its cograzing partners with a high variability (46–1570 μg/l serum). Over the period of disease, the level of the toxic metabolites (methylencyclopropylacetic acid [MCPA]‐conjugates) increased in body fluids of the AM horse (MCPA‐carnitine: Day 2, 0.246, Day 3, 0.581 μmol/l serum; MCPA‐carnitine: Day 2, 0.621, Day 3, 0.884 μmol/mmol creatinine in urine) and HGA decreased rapidly (Day 3, 2430 μg/l serum). In cograzing horses MCPA‐conjugates were not detected. HGA in seeds ranged from 268 to 367 μg/g. Although HGA was present in body fluids of healthy cograzing horses, MCPA‐conjugates were not detectable, in contrast to the AM horse. Therefore, increasing concentrations of MCPA‐conjugates are supposed to be linked with the onset of AM and both parameters seem to indicate the clinical stage of disease. However, detection of HGA in body fluids of cograzing horses might be a promising step in preventing the disease.
Publikation

Krägeloh, T.; Cavalleri, J. M. V.; Ziegler, J.; Sander, J.; Terhardt, M.; Breves, G.; Cehak, A.; Identification of hypoglycin A binding adsorbents as potential preventive measures in co-grazers of atypical myopathy affected horses Equine Vet. J. 50, 220-227, (2018) DOI: 10.1111/evj.12723

BackgroundIntestinal absorption of hypoglycin A (HGA) and its metabolism are considered major prerequisites for atypical myopathy (AM). The increasing incidence and the high mortality rate of AM urgently necessitate new therapeutic and/or preventative approaches.ObjectivesTo identify a substance for oral administration capable of binding HGA in the intestinal lumen and effectively reducing the intestinal absorption of the toxin.Study designExperimental in vitro study.MethodsSubstances commonly used in equine practice (activated charcoal composition, di‐tri‐octahedral smectite, mineral oil and activated charcoal) were tested for their binding capacity for HGA using an in vitro incubation method. The substance most effective in binding HGA was subsequently tested for its potential to reduce intestinal HGA absorption. Jejunal tissues of 6 horses were incubated in Ussing chambers to determine mucosal uptake, tissue accumulation, and serosal release of HGA in the presence and absence of the target substance. Potential intestinal metabolism in methylenecyclopropyl acetic acid (MCPA)‐conjugates was investigated by analysing their concentrations in samples from the Ussing chambers.ResultsActivated charcoal composition and activated charcoal were identified as potent HGA binding substances with dose and pH dependent binding capacity. There was no evidence of intestinal HGA metabolism.Main limitationsBinding capacity of adsorbents was tested in vitro using aqueous solutions, and in vivo factors such as transit time and composition of intestinal content, may affect adsorption capacity after oral administration.ConclusionsFor the first time, this study identifies substances capable of reducing HGA intestinal absorption. This might have major implications as a preventive measure in cograzers of AM affected horses but also in horses at an early stage of intoxication.
Publikation

Bochnia, M.; Ziegler, J.; Sander, J.; Uhlig, A.; Schaefer, S.; Vollstedt, S.; Glatter, M.; Abel, S.; Recknagel, S.; Schusser, G. F.; Wensch-Dorendorf, M.; Zeyner, A.; Hypoglycin A Content in Blood and Urine Discriminates Horses with Atypical Myopathy from Clinically Normal Horses Grazing on the Same Pasture PLOS ONE 10, e0136785, (2015) DOI: 10.1371/journal.pone.0136785

Hypoglycin A (HGA) in seeds of Acer spp. is suspected to cause seasonal pasture myopathy in North America and equine atypical myopathy (AM) in Europe, fatal diseases in horses on pasture. In previous studies, this suspicion was substantiated by the correlation of seed HGA content with the concentrations of toxic metabolites in urine and serum (MCPA-conjugates) of affected horses. However, seed sampling was conducted after rather than during an outbreak of the disease. The aim of this study was to further confirm the causality between HGA occurrence and disease outbreak by seed sampling during an outbreak and the determination of i) HGA in seeds and of ii) HGA and MCPA-conjugates in urine and serum of diseased horses. Furthermore, cograzing healthy horses, which were present on AM affected pastures, were also investigated. AM-pastures in Germany were visited to identify seeds of Acer pseudoplatanus and serum (n = 8) as well as urine (n = 6) from a total of 16 diseased horses were analyzed for amino acid composition by LC-ESI-MS/MS, with a special focus on the content of HGA. Additionally, the content of its toxic metabolite was measured in its conjugated form in body fluids (UPLC-MS/MS). The seeds contained 1.7–319.8 μg HGA/g seed. The content of HGA in serum of affected horses ranged from 387.8–8493.8 μg/L (controls < 10 μg/L), and in urine from 143.8–926.4 μg/L (controls < 10 μg/L), respectively. Healthy cograzing horses on AM-pastures showed higher serum (108.8 ± 83.76 μg/L) and urine concentrations (26.9 ± 7.39 μg/L) compared to control horses, but lower concentrations compared to diseased horses. The range of MCPA-carnitine and creatinine concentrations found in diseased horses in serum and urine were 0.17–0.65 mmol/L (controls < 0.01), and 0.34–2.05 μmol/mmoL (controls < 0.001), respectively. MCPA-glycine levels in urine of cograzing horses were higher compared to controls. Thus, the causal link between HGA intoxication and disease outbreak could be further substantiated, and the early detection of HGA in cograzing horses, which are clinically normal, might be a promising step in prophylaxis.
Publikation

Stumpe, M.; Göbel, C.; Faltin, B.; Beike, A. K.; Hause, B.; Himmelsbach, K.; Bode, J.; Kramell, R.; Wasternack, C.; Frank, W.; Reski, R.; Feussner, I.; The moss Physcomitrella patens contains cyclopentenones but no jasmonates: mutations in allene oxide cyclase lead to reduced fertility and altered sporophyte morphology New Phytol. 188, 740-749, (2010) DOI: 10.1111/j.1469-8137.2010.03406.x

Two cDNAs encoding allene oxide cyclases (PpAOC1, PpAOC2), key enzymes in the formation of jasmonic acid (JA) and its precursor (9S,13S)‐12‐oxo‐phytodienoic acid (cis‐(+)‐OPDA), were isolated from the moss Physcomitrella patens.Recombinant PpAOC1 and PpAOC2 show substrate specificity against the allene oxide derived from 13‐hydroperoxy linolenic acid (13‐HPOTE); PpAOC2 also shows substrate specificity against the allene oxide derived from 12‐hydroperoxy arachidonic acid (12‐HPETE).In protonema and gametophores the occurrence of cis‐(+)‐OPDA, but neither JA nor the isoleucine conjugate of JA nor that of cis‐(+)‐OPDA was detected.Targeted knockout mutants for PpAOC1 and for PpAOC2 were generated, while double mutants could not be obtained. The ΔPpAOC1 and ΔPpAOC2 mutants showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis.
Publikation

Pienkny, S.; Brandt, W.; Schmidt, J.; Kramell, R.; Ziegler, J.; Functional characterization of a novel benzylisoquinoline O-methyltransferase suggests its involvement in papaverine biosynthesis in opium poppy (Papaver somniferum L) Plant J. 60, 56-67, (2009) DOI: 10.1111/j.1365-313X.2009.03937.x

The benzylisoquinoline alkaloids are a highly diverse group of about 2500 compounds which accumulate in a species‐specific manner. Despite the numerous compounds which could be identified, the biosynthetic pathways and the participating enzymes or cDNAs could be characterized only for a few selected members, whereas the biosynthesis of the majority of the compounds is still largely unknown. In an attempt to characterize additional biosynthetic steps at the molecular level, integration of alkaloid and transcript profiling across Papaver species was performed. This analysis showed high expression of an expressed sequence tag (EST) of unknown function only in Papaver somniferum varieties. After full‐length cloning of the open reading frame and sequence analysis, this EST could be classified as a member of the class II type O ‐methyltransferase protein family. It was related to O ‐methyltransferases from benzylisoquinoline biosynthesis, and the amino acid sequence showed 68% identical residues to norcoclaurine 6‐O ‐methyltransferase. However, rather than methylating norcoclaurine, the recombinant protein methylated norreticuline at position seven with a K m of 44 μm using S ‐adenosyl‐l ‐methionine as a cofactor. Of all substrates tested, only norreticuline was converted. Even minor changes in the benzylisoquinoline backbone were not tolerated by the enzyme. Accordingly, the enzyme was named norreticuline 7–O ‐methyltransferase (N7OMT). This enzyme represents a novel O ‐methyltransferase in benzylisoquinoline metabolism. Expression analysis showed slightly increased expression of N7OMT in P. somniferum varieties containing papaverine, suggesting its involvement in the partially unknown biosynthesis of this pharmaceutically important compound.
Publikation

Mugford, S. G.; Yoshimoto, N.; Reichelt, M.; Wirtz, M.; Hill, L.; Mugford, S. T.; Nakazato, Y.; Noji, M.; Takahashi, H.; Kramell, R.; Gigolashvili, T.; Flügge, U.-I.; Wasternack, C.; Gershenzon, J.; Hell, R.; Saito, K.; Kopriva, S.; Disruption of Adenosine-5′-Phosphosulfate Kinase in Arabidopsis Reduces Levels of Sulfated Secondary Metabolites Plant Cell 21, 910-927, (2009) DOI: 10.1105/tpc.109.065581

Plants can metabolize sulfate by two pathways, which branch at the level of adenosine 5′-phosphosulfate (APS). APS can be reduced to sulfide and incorporated into Cys in the primary sulfate assimilation pathway or phosphorylated by APS kinase to 3′-phosphoadenosine 5′-phosphosulfate, which is the activated sulfate form for sulfation reactions. To assess to what extent APS kinase regulates accumulation of sulfated compounds, we analyzed the corresponding gene family in Arabidopsis thaliana. Analysis of T-DNA insertion knockout lines for each of the four isoforms did not reveal any phenotypical alterations. However, when all six combinations of double mutants were compared, the apk1 apk2 plants were significantly smaller than wild-type plants. The levels of glucosinolates, a major class of sulfated secondary metabolites, and the sulfated 12-hydroxyjasmonate were reduced approximately fivefold in apk1 apk2 plants. Although auxin levels were increased in the apk1 apk2 mutants, as is the case for most plants with compromised glucosinolate synthesis, typical high auxin phenotypes were not observed. The reduction in glucosinolates resulted in increased transcript levels for genes involved in glucosinolate biosynthesis and accumulation of desulfated precursors. It also led to great alterations in sulfur metabolism: the levels of sulfate and thiols increased in the apk1 apk2 plants. The data indicate that the APK1 and APK2 isoforms of APS kinase play a major role in the synthesis of secondary sulfated metabolites and are required for normal growth rates.
Publikation

Lee, C.-W.; Efetova, M.; Engelmann, J. C.; Kramell, R.; Wasternack, C.; Ludwig-Müller, J.; Hedrich, R.; Deeken, R.; Agrobacterium tumefaciens Promotes Tumor Induction by Modulating Pathogen Defense in Arabidopsis thaliana Plant Cell 21, 2948-2962, (2009) DOI: 10.1105/tpc.108.064576

Agrobacterium tumefaciens causes crown gall disease by transferring and integrating bacterial DNA (T-DNA) into the plant genome. To examine the physiological changes and adaptations during Agrobacterium-induced tumor development, we compared the profiles of salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and auxin (indole-3-acetic acid [IAA]) with changes in the Arabidopsis thaliana transcriptome. Our data indicate that host responses were much stronger toward the oncogenic strain C58 than to the disarmed strain GV3101 and that auxin acts as a key modulator of the Arabidopsis–Agrobacterium interaction. At initiation of infection, elevated levels of IAA and ET were associated with the induction of host genes involved in IAA, but not ET signaling. After T-DNA integration, SA as well as IAA and ET accumulated, but JA did not. This did not correlate with SA-controlled pathogenesis-related gene expression in the host, although high SA levels in mutant plants prevented tumor development, while low levels promoted it. Our data are consistent with a scenario in which ET and later on SA control virulence of agrobacteria, whereas ET and auxin stimulate neovascularization during tumor formation. We suggest that crosstalk among IAA, ET, and SA balances pathogen defense launched by the host and tumor growth initiated by agrobacteria.
Publikation

Fonseca, S.; Chini, A.; Hamberg, M.; Adie, B.; Porzel, A.; Kramell, R.; Miersch, O.; Wasternack, C.; Solano, R.; (+)-7-iso-Jasmonoyl-L-isoleucine is the endogenous bioactive jasmonate Nat. Chem. Biol. 5, 344-350, (2009) DOI: 10.1038/nchembio.161

Hormone-triggered activation of the jasmonate signaling pathway in Arabidopsis thaliana requires SCFCOI1-mediated proteasome degradation of JAZ repressors. (−)-JA-L-Ile is the proposed bioactive hormone, and SCFCOI1 is its likely receptor. We found that the biological activity of (−)-JA-L-Ile is unexpectedly low compared to coronatine and the synthetic isomer (+)-JA-L-Ile, which suggests that the stereochemical orientation of the cyclopentanone-ring side chains greatly affects receptor binding. Detailed GC-MS and HPLC analyses showed that the (−)-JA-L-Ile preparations currently used in ligand binding studies contain small amounts of the C7 epimer (+)-7-iso-JA-L-Ile. Purification of each of these molecules demonstrated that pure (−)-JA-L-Ile is inactive and that the active hormone is (+)-7-iso-JA-L-Ile, which is also structurally more similar to coronatine. In addition, we show that pH changes promote conversion of (+)-7-iso-JA-L-Ile to the inactive (−)-JA-L-Ile form, thus providing a simple mechanism that can regulate hormone activity through epimerization.
Publikation

Ziegler, J.; Facchini, P. J.; Geißler, R.; Schmidt, J.; Ammer, C.; Kramell, R.; Voigtländer, S.; Gesell, A.; Pienkny, S.; Brandt, W.; Evolution of morphine biosynthesis in opium poppy Phytochemistry 70, 1696-1707, (2009) DOI: 10.1016/j.phytochem.2009.07.006

Benzylisoquinoline alkaloids (BIAs) are a group of nitrogen-containing plant secondary metabolites comprised of an estimated 2500 identified structures. In BIA metabolism, (S)-reticuline is a key branch-point intermediate that can be directed into several alkaloid subtypes with different structural skeleton configurations. The morphinan alkaloids are one subclass of BIAs produced in only a few plant species, most notably and abundantly in the opium poppy (Papaver somniferum). Comparative transcriptome analysis of opium poppy and several other Papaver species that do not accumulate morphinan alkaloids showed that known genes encoding BIA biosynthetic enzymes are expressed at higher levels in P. somniferum. Three unknown cDNAs that are co-ordinately expressed with several BIA biosynthetic genes were identified as enzymes in the pathway. One of these enzymes, salutaridine reductase (SalR), which is specific for the production of morphinan alkaloids, was isolated and heterologously overexpressed in its active form not only from P. somniferum, but also from Papaver species that do not produce morphinan alkaloids. SalR is a member of a class of short chain dehydrogenase/reductases (SDRs) that are active as monomers and possess an extended amino acid sequence compared with classical SDRs. Homology modelling and substrate docking revealed the substrate binding site for SalR. The amino acids residues conferring salutaridine binding were compared to several members of the SDR family from different plant species, which non-specifically reduce (−)-menthone to (+)-neomenthol. Previously, it was shown that some of these proteins are involved in plant defence. The recruitment of specific monomeric SDRs from monomeric SDRs involved in plant defence is discussed.
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