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Publikation

Ziegler, J.; Bochnia, M.; Zeyner, A.; Aminosäurennachweis in geringsten ProbenmengenBestimmung von Hypoglycin A Wiley Analytical Science (2021)

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Publikation

Bochnia, M.; Ziegler, J.; Glatter, M.; Zeyner, A.; Hypoglycin A in cow’s milk—A pilot study Toxins 13, 381, (2021) DOI: 10.3390/toxins13060381

Hypoglycin A (HGA) originating from soapberry fruits (litchi, and ackee) seeds or seedlings from the sycamore maple (SM) tree (related to Sapindaceae) may cause Jamaican vomiting sickness in humans and atypical myopathy in horses and ruminants. A possible transfer into dairy cow’s milk cannot be ruled out since the literature has revealed HGA in the milk of mares and in the offal of captured deer following HGA intoxication. From a study, carried out for another purpose, bulk raw milk samples from four randomly selected dairy farms were available. The cows were pastured in the daytime. A sycamore maple tree was found on the pasture of farm No. 1 only. Bulk milk from the individual tank or milk filling station was sampled in parallels and analyzed for HGA by LC-ESI-MS/MS. Measurable concentrations of HGA occurred only in milk from farm No. 1 and amounted to 120 and 489 nmol/L. Despite low and very variable HGA concentrations, the results indicate that the ingested toxin, once eaten, is transferred into the milk. However, it is unknown how much HGA the individual cow ingested during grazing and what amount was transferred into the bulk milk samples. As a prerequisite for a possible future safety assessment, carry-over studies are needed. Furthermore, the toxins’ stability during milk processing should also be investigated as well.
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

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

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

Danon, A.; Miersch, O.; Felix, G.; op den Camp, R. G. L.; Apel, K.; Concurrent activation of cell death-regulating signaling pathways by singlet oxygen in Arabidopsis thaliana Plant J. 41, 68-80, (2005) DOI: 10.1111/j.1365-313X.2004.02276.x

Upon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen (1O2), a non‐radical reactive oxygen species that is restricted to the plastid compartment. Immediately after the shift, plants stop growing and develop necrotic lesions. We have established a protoplast system, which allows detection and characterization of the death response in flu induced by the release of 1O2. Vitamin B6 that quenches 1O2 in fungi was able to protect flu protoplasts from cell death. Blocking ethylene production was sufficient to partially inhibit the death reaction. Similarly, flu mutant seedlings expressing transgenic NahG were partially protected from the death provoked by the release of 1O2, indicating a requirement for salicylic acid (SA) in this process, whereas in cells depleted of both, ethylene and SA, the extent of cell death was reduced to the wild‐type level. The flu mutant was also crossed with the jasmonic acid (JA)‐depleted mutant opr3 , and with the JA, OPDA and dinor OPDA (dnOPDA)‐depleted dde2‐2 mutant. Analysis of the resulting double mutants revealed that in contrast to the JA‐induced suppression of H2O2/superoxide‐dependent cell death reported earlier, JA promotes singlet oxygen‐mediated cell death in flu , whereas other oxylipins such as OPDA and dnOPDA antagonize this death‐inducing activity of JA.
Publikation

Nibbe, M.; Hilpert, B.; Wasternack, C.; Miersch, O.; Apel, K.; Cell death and salicylate- and jasmonate-dependent stress responses in Arabidopsis are controlled by single cet genes Planta 216, 120-128, (2002) DOI: 10.1007/s00425-002-0907-1

The jasmonic acid (JA)-dependent regulation of the Thi2.1 gene had previously been exploited for setting up a genetic screen for the isolation of signal transduction mutants of Arabidopsis thaliana (L.) Heynh. that constitutively express the thionin gene. Several cet mutants had been isolated which showed a constitutive expression of the thionin gene. These cet mutants, except for one, also showed spontaneous leaf cell necrosis and were up-regulated in the expression of the PR1 gene, reactions often associated with the systemic acquired resistance (SAR) pathway. Four of these cet mutants, cet1, cet2, cet3 and cet4.1 were crossed with the fad triple and coi1 mutants that are blocked at two steps within the JA-dependent signaling pathway, and with transgenic NahG plants that are deficient in salicylic acid (SA) and are unable to activate SAR. Analysis of the various double-mutant lines revealed that the four cet genes act within a signaling cascade at or prior to branch points from which not only JA-dependent signals but also SA-dependent signaling and cell death pathways diverge.
Publikation

Hilpert, B.; Bohlmann, H.; Den Camp, R. o.; Przybyla, D.; Miersch, O.; Buchala, A.; Apel, K.; Isolation and characterization of signal transduction mutants of Arabidopsis thaliana that constitutively activate the octadecanoid pathway and form necrotic microlesions Plant J. 26, 435-446, (2001) DOI: 10.1046/j.1365-313X.2001.2641036.x

Thionins are a group of antimicrobial polypeptides that form part of the plant's defense mechanism against pathogens. The Thi 2.1 thionin gene of Arabidopsis thaliana has been shown to be inducible by jasmonic acid (JA), an oxylipin‐like hormone derived from oxygenated linolenic acid and synthesized via the octadecanoid pathway. The JA‐dependent regulation of the Thi 2.1 gene has been exploited for setting up a genetic screen for the isolation of signal transduction mutants that constitutively express the Thi 2.1 gene. Ten cet‐mutants have been isolated which showed a c onstitutive e xpression of the t hionin gene. Allelism tests revealed that they represent at least five different loci. Some mutants are dominant, others recessive, but all cet mutations behaved as monogenic traits when backcrossed with Thi 2.1‐GUS plants. Some of the mutants overproduce JA and its bioactive precursor 12‐oxophytodienoic acid (OPDA) up to 40‐fold while others have the same low levels as the control wildtype plants. Two of the mutants showed a strong induction of both the salicylic acid (SA)‐ and the JA‐dependent signaling pathways, while the majority seems to be affected only in the octadecanoid pathway. The Thi 2.1 thionin gene and the Pdf 1.2 defensin gene are activated independently, though both are regulated by JA. The cet‐mutants, except for one, also show a spontaneous leaf cell necrosis, a reaction often associated with the systemic acquired resistance (SAR) pathway.
Publikation

Ziegler, J.; Stenzel, I.; Hause, B.; Maucher, H.; Hamberg, M.; Grimm, R.; Ganal, M.; Wasternack, C.; Molecular Cloning of Allene Oxide Cyclase J. Biol. Chem. 275, 19132-19138, (2000) DOI: 10.1074/jbc.M002133200

Allene oxide cyclase (EC 5.3.99.6) catalyzes the stereospecific cyclization of an unstable allene oxide to (9S,13S)-12-oxo-(10,15Z)-phytodienoic acid, the ultimate precursor of jasmonic acid. This dimeric enzyme has previously been purified, and two almost identical N-terminal peptides were found, suggesting allene oxide cyclase to be a homodimeric protein. Furthermore, the native protein was N-terminally processed. Using degenerate primers, a polymerase chain reaction fragment could be generated from tomato, which was further used to isolate a full-length cDNA clone of 1 kilobase pair coding for a protein of 245 amino acids with a molecular mass of 26 kDa. Whereas expression of the whole coding region failed to detect allene oxide cyclase activity, a 5′-truncated protein showed high activity, suggesting that additional amino acids impair the enzymatic function. Steric analysis of the 12-oxophytodienoic acid formed by the recombinant enzyme revealed exclusive (>99%) formation of the 9S,13Senantiomer. Exclusive formation of this enantiomer was also found in wounded tomato leaves. Southern analysis and genetic mapping revealed the existence of a single gene for allene oxide cyclase located on chromosome 2 of tomato. Inspection of the N terminus revealed the presence of a chloroplastic transit peptide, and the location of allene oxide cyclase protein in that compartment could be shown by immunohistochemical methods. Concomitant with the jasmonate levels, the accumulation of allene oxide cyclase mRNA was transiently induced after wounding of tomato leaves.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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