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Bioassay-guided fractionation of extracts from Easter lily (Lilium longiflorum) flowers reveals unprecedented structural variability of steroidal glycoalkaloids.
Toxicon
PUBLISHED: 05-29-2014
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Several Lilium species are nephrotoxic in cats (Felis silvestris catus), among them Easter lilies (Lilium longiflorum). Although clinical trials have been carried out, the causative toxic phytochemicals have not yet been identified. We thus aimed to determine the toxic constituents of Easter lily flowers applying a bioassay-guided approach based on a feline kidney cell line model. The bioassay-guided fractionation traced the observed cytotoxicity to a complex mixture of compounds that were tentatively identified as steroidal glycoalkaloids of the solasodine-type, based on multiple-fragmentation ion trap and high-resolution mass spectrometry. The glycoalkaloids in the active fraction possessed trisaccharide chains, and at least 16 different congeners could be separated using liquid chromatography-mass spectrometry. The two principal compounds were solasodine trisaccharides containing two hexose and one deoxy-hexose unit. In the remaining 14 analogues, one or two of the hydroxyl groups of the second hexose from the aglycone were acetylated. In addition, some of the analogues appeared to be carbonate esters. Esterification of steroidal glycoalkaloids in plants has only been reported once and was in accordance with higher antifungal activity of the acetylated versus the parent congener. Our pilot study shows that esterification of steroidal glycoalkaloids in Lilium species might be common resulting in an array of different analogues with largely unexplored structural variability and bioactivity.
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Indole-diterpenoid profiles of Claviceps paspali and Claviceps purpurea from high-resolution Fourier transform Orbitrap mass spectrometry.
Rapid Commun. Mass Spectrom.
PUBLISHED: 02-28-2014
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The biological activities most commonly associated with indole-diterpenoids are tremorgenicity in mammals and toxicity in insects through modulation of ion channels. The neurotoxic effects of some analogues are the cause of syndromes such as 'ryegrass staggers' and 'Paspalum staggers' in cattle and sheep. Our purpose was to obtain and interpret mass spectra of some pure Claviceps-related indole-diterpenoids (paspaline, paspalinine, paxilline, paspalitrems A and B) to facilitate identification of related compounds for which standards were not available.
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Faces of a changing climate: semi-quantitative multi-mycotoxin analysis of grain grown in exceptional climatic conditions in norway.
Toxins (Basel)
PUBLISHED: 08-22-2013
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Recent climatological research predicts a significantly wetter climate in Southern Norway as a result of global warming. Thus, the country has already experienced unusually wet summer seasons in the last three years (2010-2012). The aim of this pilot study was to apply an existing multi-analyte LC-MS/MS method for the semi-quantitative determination of 320 fungal and bacterial metabolites in Norwegian cereal grain samples from the 2011 growing season. Such knowledge could provide important information for future survey and research programmes in Norway. The method includes all regulated and well-known mycotoxins such as aflatoxins, trichothecenes, ochratoxin A, fumonisins and zearalenone. In addition, a wide range of less studied compounds are included in the method, e.g., Alternaria toxins, ergot alkaloids and other metabolites produced by fungal species within Fusarium, Penicillium and Aspergillus. Altogether, 46 metabolites, all of fungal origin, were detected in the 76 barley, oats and wheat samples. The analyses confirmed the high prevalence and relatively high concentrations of type-A and -B trichothecenes (e.g., deoxynivalenol up to 7230 µg/kg, HT-2 toxin up to 333 µg/kg). Zearalenone was also among the major mycotoxins detected (maximum concentration 1670 µg/kg). Notably, several other Fusarium metabolites such as culmorin, 2-amino-14,16-dimethyloctadecan-3-ol and avenacein Y were co-occurring. Furthermore, the most prevalent Alternaria toxin was alternariol with a maximum concentration of 449 µg/kg. A number of Penicillium and Aspergillus metabolites were also detected in the samples, e.g., sterigmatocystin in concentrations up to 20 µg/kg.
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Urinary analysis reveals high deoxynivalenol exposure in pregnant women from Croatia.
Food Chem. Toxicol.
PUBLISHED: 05-24-2013
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In this pilot survey the levels of various mycotoxin biomarkers were determined in third trimester pregnant women from eastern Croatia. First void urine samples were collected and analysed using a "dilute and shoot" LC-ESI-MS/MS multi biomarker method. Deoxynivalenol (DON) and its metabolites: deoxynivalenol-15-glucuronide and deoxynivalenol-3-glucuronide were detected in 97.5% of the studied samples, partly at exceptionally high levels, while ochratoxin A was found in 10% of the samples. DON exposure was primarily reflected by the presence of deoxynivalenol-15-glucuronide with a mean concentration of 120?gL(-1), while free DON was detected with a mean concentration of 18.3?gL(-1). Several highly contaminated urine samples contained a third DON conjugate, tentatively identified as deoxynivalenol-7-glucuronide by MS/MS scans. The levels of urinary DON and its metabolites measured in this study are the highest ever reported, and 48% of subjects were estimated to exceed the provisional maximum tolerable daily intake (1?gkg(-1)b.w.).
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Enzyme-assisted synthesis and structural characterization of the 3-, 8-, and 15-glucuronides of deoxynivalenol.
J. Agric. Food Chem.
PUBLISHED: 02-18-2013
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4-Deoxynivalenol is one of the most prevalent mycotoxins in grain-based food and feed products worldwide. Conjugation of deoxynivalenol to glucuronic acid and elimination via the urine appears to be the major metabolism pathway, although with differing efficiency in different species. In order to make pure deoxynivalenol glucuronides for analytical methodologies available we intended to enzymatically synthesize glucuronides of deoxynivalenol using rat and human liver microsomes supplemented with uridine 5-diphosphoglucuronic acid and alamethicin as detergent. Three glucuronides were isolated and purified using solid-phase extraction of microsomal incubations and subsequent semipreparative hydrophilic interaction chromatography. NMR spectra were obtained for all three compounds from solutions in methanol, showing that deoxynivalenol 3-O-?-D-glucuronide and deoxynivalenol 15-O-?-D-glucuronide were the major products from incubations of deoxynivalenol with rat and human liver microsomes, respectively. The NMR spectra of a third glucuronide showed replacement of the C-8 carbonyl by a ketal carbon. This glucuronide was finally identified as deoxynivalenol 8-O-?-D-glucuronide. The present study provides unequivocal structural evidence for three glucuronides of deoxynivalenol formed by liver enzymes.
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Cytosol protein regulation in H295R steroidogenesis model induced by the zearalenone metabolites, ?- and ?-zearalenol.
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PUBLISHED: 09-01-2011
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?- and ?-zearalenol (?-ZOL and ?-ZOL, respectively) are metabolites of the mycotoxin zearalenone (ZEN). All three individual mycotoxins have shown to be biological active i.e. being estrogenic and able to stimulate cellular proliferation albeit at different strengths. In this work, cytosol protein expression was determined by using stable-isotope labelling by amino acids in cell culture (SILAC) upon exposure of ?-ZOL and ?-ZOL to the steroidogenesis cell model H295R. A total of 14 and 5 individual proteins were found to be significantly regulated by ?-ZOL and ?-ZOL, respectively. Interestingly, there were no common protein regulations by the metabolites or the parent mycotoxin ZEN. Furthermore, the regulated proteins were assigned to networks and groups of actions that also differed from one another suggesting that the three individual mycotoxins may have unique biological activities.
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In vitro metabolism of the mycotoxin enniatin B in different species and cytochrome p450 enzyme phenotyping by chemical inhibitors.
Drug Metab. Dispos.
PUBLISHED: 05-26-2011
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Enniatins are cyclic hexapeptidic mycotoxins produced by fungi growing on field grains, especially in wet climates. They show considerable resistance to food and feed processing technologies and might cause intoxication of humans and animals. Enniatins are also under exploration as anticancer drugs. The observed difference of in vitro and in vivo toxicities suggests low absorption or fast elimination of the enniatins after oral uptake. In the study presented here, in vitro metabolism studies of enniatin B were performed using rat, dog, and human liver microsomes under conditions of linear kinetics to estimate the respective elimination rates. Furthermore, cytochrome P450 reaction phenotyping with chemical inhibitors selective for human enzymes was carried out. Twelve metabolites were separated and characterized by multiple high-performance liquid chromatographic/mass spectrometric analyses as products of oxidation and demethylation reactions. Biotransformation rates and metabolite patterns varied considerably in the three species. The intrinsic clearances determined in assays with rat, dog, and human liver microsomes were 1.16, 8.23, and 1.13 l/(h · kg), respectively. The predicted enniatin B in vivo blood clearances were 1.57 l/(h · kg) in rats, 1.67 l/(h · kg) in dogs, and 0.63 l/(h · kg) in humans. CYP3A4 was important for enniatin B metabolism in human microsomes as shown by 80% inhibition and impaired metabolite formation in the presence of troleandomycin. CYP1A2 and CYP2C19 were additionally involved. Preliminary results showed that CYP3A and CYP1A might also be relevant in rats and dogs. The extensive hepatic metabolism could explain the reduced in vivo potential of enniatin B.
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In vitro phase I metabolism of the depsipeptide enniatin B.
Anal Bioanal Chem
PUBLISHED: 02-17-2011
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The enniatins are a group of more than 20 cyclic depsipeptides from fungi with numerous biological effects. Enniatin B is commonly one of the principal analogues in species of the genus Fusarium, known to have ionophoric, antibiotic and insecticidal activity. In the present study, enniatin B was incubated with rat, dog and human liver microsomes. The compound was extensively metabolised, and 12 biotransformation products (M1-M12) were detected and their structures tentatively identified using a combination of mass spectrometric techniques and chemical derivatisation. Ion trap mass spectrometry, multiple-stage MS(n) fragmentation and high-resolution mass spectrometry were the instrumental backbone for structural determination, while acetylation, methylation and Jones oxidation were useful derivatisation techniques for the localisation of the site of biotransformation. Comparison of mass spectrometric data of the metabolism products with that of enniatin B suggested that M1-M5 are monohydroxylated species, while M8-M12 are the result of multiple oxidations (oxygenation and dehydrogenation). Metabolites M6 and M7 appeared to be enniatin B homologues and are the result of N-demethylation. Our findings show that oxidation and N-demethylation are the principal metabolic pathways in enniatin B phase I metabolism.
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Isolation and structure elucidation of secopenitrem D, an indole alkaloid from Penicillium crustosum Thom.
Toxicon
PUBLISHED: 09-07-2010
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Chemical investigation of three isolates of Penicillium crustosum Thom cultures, one of which was derived from a recent dog poisoning investigation, has led to the isolation and structure elucidation of secopenitrem D (1). Penitrems A-F and roquefortine C were also present in the isolates analyzed. The structure of 1 was unambiguously assigned based on extensive 1D and 2D-NMR spectroscopic experiments, MS-aided structural studies and by comparison with structurally related congeners. Secopenitrem D lacks the C-16-C-18 ether linkage present in penitrems A-F.
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Electron ionisation mass spectrometry of the pentafluoropropionate esters of trichothecene analogues and culmorin compounds from Fusarium species.
Rapid Commun. Mass Spectrom.
PUBLISHED: 08-27-2010
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This paper reports the mass spectra, obtained after electron ionisation (EI) at 70 eV, of 34 trichothecenes and five culmorin compounds after acylation with pentafluoropropionic anhydride. The derivatised fungal metabolites were separated by gas chromatography, and the mass spectra were obtained by scanning of a single quadrupole mass filter in the scan range m/z 200-900. The fragmentation pathways of three trichothecenes (triacetyl-deoxynivalenol, 4,15-diacetoxy-scirpenol, T-2 toxin) have been studied in more detail by linked scan-high-resolution mass spectrometry. The most common trichothecenes are today more often routinely analysed using LC/MS-based methodologies. However, EI-MS may give complementary structural information, and the data that are summarised in this article may help to identify analogues of one of the largest class of mycotoxins, the tricothecenes, as well as culmorin compounds that are commonly co-produced by Fusarium culmorum and F. graminearum in cultures or naturally contaminated samples.
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Phomenins and fatty acids from Alternaria infectoria.
Toxicon
PUBLISHED: 01-06-2010
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Taxa of the Alternaria infectoria species group are the predominant Alternaria spp. found in cereals in Northern Europe. While several pyrones have been isolated from A. infectoria and described as taxonomical markers for species identification, information about the bioactivity of metabolites from the fungus is missing. Bioassay-guided fractionation of rice culture extracts from several strains of A. infectoria linked the observed toxicity of the extracts in MRC-5 cells to free fatty acids, i.e. linoleic acid and alpha-linolenic acid. The fungus also produced a cytotoxic pyrone, which upon isolation and NMR spectroscopic analysis was identified as a mixture of phomenins A and B (approximately 10:1), which have not previously been isolated from an Alternaria species.
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Indole-diterpenes and ergot alkaloids in Cynodon dactylon (Bermuda grass) infected with Claviceps cynodontis from an outbreak of tremors in cattle.
J. Agric. Food Chem.
PUBLISHED: 11-07-2009
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Tremorgenic syndromes in mammals are commonly associated with indole-diterpenoid alkaloids of fungal origin. Cattle are sometimes affected by tremors (also called "staggers") when they graze on toxic grass pastures, and Bermuda grass ( Cynodon dactylon , kweek) has been known to be associated with tremors for several decades. This study reports the identification of paspalitrems and paspaline-like indole-diterpenes in the seedheads of Claviceps cynodontis -infected Bermuda grass collected from a pasture that had caused a staggers syndrome in cattle in South Africa and thereby links the condition to specific mycotoxins. The highest concentration (about 150 mg/kg) was found for paspalitrem B. Ergonovine and ergine (lysergic acid amide), together with their C-8 epimers, were found to co-occur with the indole-diterpenes at concentrations of about 10 microg/kg. The indole-diterpene profile of the extract from the ergotized Bermuda grass was similar to that of Claviceps paspali sclerotia. However, the C. paspali sclerotia contained in addition agroclavine and elymoclavine. This is the first study linking tremors associated with grazing of Bermuda grass to specific tremorgenic indole-diterpenoid mycotoxins.
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Structural studies on minor enniatins from Fusarium sp. VI 03441: novel N-methyl-threonine containing enniatins.
Toxicon
PUBLISHED: 02-06-2009
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A strain of a yet unidentified Fusarium sp. produced in addition to enniatins A1, B and B1 a number of minor enniatins. The strain formed a well supported clade with strains identified as Fusarium acuminatum (Gibberella acuminata) in phylogenetic analyses using the TEF-1alpha gene sequences. Two of the minor enniatins were easily recognised as hydroxylated species on the basis of their fragment ion spectra. The hydroxylation could be traced to one of the amino acid moieties using multiple-stage ion trap mass spectrometry. Different approaches for acetylation of the isolated compounds and complete hydrolysis supported the elucidation of the amino acid moiety as 3-hydroxy-2-methylamino-butyric acid, which is equivalent with N-methyl-threonine. The primary structures of the two enniatins were tentatively determined to be cyclo[Hiv-N-Me-Val-Hiv-N-Me-Val-Hiv-N-Me-Thr] and cyclo[Hiv-N-Me-Leu-Hiv-N-Me-Val-Hiv-N-Me-Thr]. The two depsipeptides represent new analogues and were named enniatin P1 and P2, respectively.
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Identification of early fumonisin biosynthetic intermediates by inactivation of the FUM6 gene in Fusarium verticillioides.
J. Agric. Food Chem.
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Fumonisins are polyketide mycotoxins produced by the maize pathogen Fusarium verticillioides and are associated with multiple human and animal diseases. A fumonisin biosynthetic pathway has been proposed, but structures of early pathway intermediates have not been demonstrated. The F. verticillioides FUM6 gene is required for an early pathway step. Here, metabolites produced by strains of the fungus with an inactivated FUM6 gene were purified and shown by mass spectrometry and NMR spectroscopy to have fumonisin-like structures but without substitutions at C-14 and C-15. The major metabolite was 2-amino-12,16-dimethylicosane-3,10-diol. Lesser amounts of 3-keto and triol analogues of the metabolite were also identified. In precursor feeding experiments, 2-amino-12,16-dimethylicosane-3,10-diol was transformed to fumonisins by a F. verticillioides strain with an inactive fumonisin polyketide synthase gene. The results support the hypothesis that the FUM6-encoded enzyme catalyzes fumonisin C-14 and C-15 hydroxylation and provide direct spectroscopic and biochemical evidence for structures of early intermediates in fumonisin biosynthesis.
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