Cycad Seeds Research Articles

Excitatory amino acid beta-N-methylamino-L-alanine is a putative environmental neurotoxin

The amino acid beta-N-methylamino-L-alanine (L-BMAA) has been associated with the amyotrophic lateral sclerosis/parkinsonismdementia complex in three distinct western Pacific populations. The putative neurotoxin is produced by cyanobacteria, which live symbiotically in the roots of cycad trees. L-BMAA was thought to be a threat only to those few populations whose diet and medicines rely heavily on cycad seeds. However, the recent discovery that cyanobacteria from diverse terrestrial, freshwater, and saltwater ecosystems around the world produce the toxin requires a reassessment of whether it poses a larger health threat. Therefore, it is proposed that monitoring L-BMAA levels in cyanobacteria-contaminated water supplies might be prudent.

Prominent human health impacts from several marine microbes: history, ecology, and public health implications.

This paper overviews several examples of important public health impacts by marine microbes and directs readers to the extensive literature germane to these maladies. These examples include three types of dinoflagellates (Gambierdiscus spp., Karenia brevis, and Alexandrium fundyense), BMAA-producing cyanobacteria, and infectious microbes. The dinoflagellates are responsible for ciguatera fish poisoning, neurotoxic shellfish poisoning, and paralytic shellfish poisoning, respectively, that have plagued coastal populations over time. Research interest on the potential for marine cyanobacteria to contribute BMAA into human food supplies has been derived by BMAA's discovery in cycad seeds and subsequent implication as the putative cause of amyotrophic lateral sclerosis/parkinsonism dementia complex among the Chamorro people of Guam. Recent UPLC/MS analyses indicate that recent reports that BMAA is prolifically distributed among marine cyanobacteria at high concentrations may be due to analyte misidentification in the analytical protocols being applied for BMAA. Common infectious microbes (including enterovirus, norovirus, Salmonella, Campylobacter, Shigella, Staphylococcus aureus, Cryptosporidium, and Giardia) cause gastrointestinal and skin-related illness. These microbes can be introduced from external human and animal sources, or they can be indigenous to the marine environment.

In Vitro Effects of Cholesterol β-d-Glucoside, Cholesterol and Cycad Phytosterol Glucosides on Respiration and Reactive Oxygen Species Generation in Brain Mitochondria

The cluster of neurodegenerative disorders in the western Pacific termed amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC) has been repeatedly linked to the use of seeds of various species of cycad. Identification and chemical synthesis of the most toxic compounds in the washed cycad seeds, a variant phytosteryl glucosides, and even more toxic cholesterol β-D-glucoside (CG), which is produced by the human parasite Helicobacter pylori, provide a possibility to study in vitro the mechanisms of toxicity of these compounds. We studied in detail the effects of CG on the respiratory activities and generation of reactive oxygen species (ROS) by nonsynaptic brain and heart mitochondria oxidizing various substrates. The stimulatory effects of CG on respiration and ROS generation showed strong substrate dependence, suggesting involvement of succinate dehydrogenase (complex II). Maximal effects on ROS production were observed with 1 μmol CG/1 mg mitochondria. At this concentration the cycad toxins β-sitosterol-β-D-glucoside and stigmasterol-β-D-glucoside had effects on respiration and ROS production similar to CG. However, poor solubility precluded full concentration analysis of these toxins. Cholesterol, stigmasterol and β-sitosterol had no effect on mitochondrial functions studied at concentrations up to 100 μmol/mg protein. Our results suggest that CG may influence mitochondrial functions through changes in the packing of the bulk membrane lipids, as was shown earlier by Deliconstantinos et al. (Biochem Cell Biol 67:16-24, 1989). The neurotoxic effects of phytosteryl glucosides and CG may be associated with increased oxidative damage of neurons. Unlike heart mitochondria, in activated neurons mitochondria specifically increase ROS production associated with succinate oxidation (Panov et al., J Biol Chem 284:14448-14456, 2009).

Caleosin serves as the major structural protein as efficient as oleosin on the surface of seed oil bodies

Oleosin, the most abundant protein in oil bodies of all examined angiosperm seeds, has been demonstrated to serve as a structural protein to maintain the integrity of these lipid storage organelles. Caleosin, a minor protein in oil bodies of angiosperm seeds, is assumed to anchor to the organelles in a manner similar to oleosin, i.e., via its central hydrophobic domain with a unique proline-knot motif presumably responsible for the targeting to nascent oil bodies. Recently, we found that stable oil bodies in cycad seeds were mainly sheltered by caleosin in the absence of oleosin. This finding suggests that caleosin is competent as the major structural protein, just like oleosin, on the surface of seed oil bodies. A search of literature shows that genes putatively encoding for caleosin-, but not oleosin-like proteins, are present in more primitive species, such as algae and fungi. We hypothesize that oleosin may have been derived from caleosin after a long term of divergent evolution.

Liquid chromatographic determination of the cyanobacterial toxin beta-n-methylamino-L-alanine in algae food supplements, freshwater fish, and bottled water.

Beta-N-Methylamino-L-alanine (BMAA) is a neurotoxin originally found in cycad seeds and now known to be produced by many species of freshwater and marine cyanobacteria. We developed a method for its determination in blue-green algae (BGA) food supplements, freshwater fish, and bottled water by using a strong cation-exchange, solid-phase extraction column for cleanup after 0.3 M trichloroacetic acid extraction of BGA supplements and fish. Bottled water was applied directly onto the solid-phase extraction column. For analysis of carbonated water, sonication and pH adjustment to 1.5 were needed. To determine protein-bound BMAA, the protein pellet left after extraction of the BGA supplement and fish was hydrolyzed by boiling with 6 M hydrochloric acid; BMAA was cleaned up on a C18 column and a strong cation-exchange, solid-phase extraction column. Determination of BMAA was by liquid chromatography of the fluorescent derivative formed with 9-fluorenylmethyl chloroformate. The method was validated by recovery experiments using spiking levels of 1.0 to 10 microg/g for BGA supplements, 0.5 to 5.0 microg/g for fish, and 0.002 microg/g for bottled water; mean recoveries were in the range of 67 to 89% for BGA supplements and fish, and 59 to 92% for bottled water. Recoveries of BMAA from spiked extracts of hydrolyzed protein from BGA supplements and fish ranged from 66 to 83%. The cleanup developed provides a useful method for surveying foods and supplements for BMAA and protein-bound BMAA.

The role of environmental mercury, lead and pesticide exposure in development of amyotrophic lateral sclerosis

Exposure to an environmental toxicant as a risk factor in the development of amyotrophic lateral sclerosis (ALS) was first hinted at (demonstrated) in the Chamorro indigenous people of Guam. During the 1950s and 1960s these indigenous people presented an extremely high incidence of ALS which was presumed to be associated with the consumption of flying fox and cycad seeds. No other strong association between ALS and environmental toxicants has since been reported, although circumstantial epidemiological evidence has implicated exposure to heavy metals such as lead and mercury, industrial solvents and pesticides especially organophosphates and certain occupations such as playing professional soccer. Given that only ∼10% of all ALS diagnosis have a genetic basis, a gene–environmental interaction provides a plausible explanation for the other 90% of cases. This mini-review provides an overview of our current knowledge of environmental etiologies of ALS with emphasis on the effects of mercury, lead and pesticides as potential risk factors in developing ALS. Epidemiologic and experimental evidence from animal models investigating the possible association between exposure to environmental toxicant and ALS disease has proven inconclusive. Nonetheless, there are indications that there may be causal links, and a need for more research.

The chitin‐binding capability of Cy‐AMP1 from cycad is essential to antifungal activity

Antimicrobial peptides are important components of the host innate immune responses by exerting broad-spectrum microbicidal activity against pathogenic microbes. Cy-AMP1 found in the cycad (Cycas revoluta) seeds has chitin-binding ability, and the chitin-binding domain was conserved in knottin-type and hevein-type antimicrobial peptides. The recombinant Cy-AMP1 was expressed in Escherichia coli and purified to study the role of chitin-binding domain. The mutants of Cy-AMP1 lost chitin-binding ability completely, and its antifungal activity was markedly decreased in comparison with native Cy-AMP1. However, the antimicrobial activities of the mutant peptides are nearly identical to that of native one. It was suggested that the chitin-binding domain plays an essential role in antifungal, but not antimicrobial, activity of Cy-AMP1.

Testing co‐evolutionary hypotheses over geological timescales: interactions between Mesozoic non‐avian dinosaurs and cycads

The significance of co-evolution over ecological timescales is well established, yet it remains unclear to what extent co-evolutionary processes contribute to driving large-scale evolutionary and ecological changes over geological timescales. Some of the most intriguing and pervasive long-term co-evolutionary hypotheses relate to proposed interactions between herbivorous non-avian dinosaurs and Mesozoic plants, including cycads. Dinosaurs have been proposed as key dispersers of cycad seeds during the Mesozoic, and temporal variation in cycad diversity and abundance has been linked to dinosaur faunal changes. Here we assess the evidence for proposed hypotheses of trophic and evolutionary interactions between these two groups using diversity analyses, a new database of Cretaceous dinosaur and plant co-occurrence data, and a geographical information system (GIS) as a visualisation tool. Phylogenetic evidence suggests that the origins of several key biological properties of cycads (e.g. toxins, bright-coloured seeds) likely predated the origin of dinosaurs. Direct evidence of dinosaur-cycad interactions is lacking, but evidence from extant ecosystems suggests that dinosaurs may plausibly have acted as seed dispersers for cycads, although it is likely that other vertebrate groups (e.g. birds, early mammals) also played a role. Although the Late Triassic radiations of dinosaurs and cycads appear to have been approximately contemporaneous, few significant changes in dinosaur faunas coincide with the late Early Cretaceous cycad decline. No significant spatiotemporal associations between particular dinosaur groups and cycads can be identified - GIS visualisation reveals disparities between the spatiotemporal distributions of some dinosaur groups (e.g. sauropodomorphs) and cycads that are inconsistent with co-evolutionary hypotheses. The available data provide no unequivocal support for any of the proposed co-evolutionary interactions between cycads and herbivorous dinosaurs - diffuse co-evolutionary scenarios that are proposed to operate over geological timescales are plausible, but such hypotheses need to be firmly grounded on direct evidence of interaction and may be difficult to support given the patchiness of the fossil record.

BMAA – an unusual cyanobacterial neurotoxin

Abstract The toxin ss-N-methylamino-L-alanine (BMAA) was proposed to contribute to the ALS/Parkinsonism-dementia complex of Guam (ALS/PDC) based on its presence in cycad seeds, which constituted a dietary item in afflicted populations, and its ability to induce a similar disease phenotype in primates. Although the role of BMAA in human neurodegenerative disease is still highly debated, it appears to injure cultured neurons via mechanisms involving overactivation of neuroexcitatory glutamate receptors. However, BMAA lacks the side-chain acidic group of glutamate and other excitatory amino acids, and in its place has an amino group. In past studies we found that toxic and excitatory effects of BMAA on cultured neurons were dependent upon the presence of bicarbonate in the medium, and suggested that formation of a carbamate adduct of the side-chain amino group might produce structures capable of activating glutamate receptors. Also, while BMAA is a weal agonist at NMDA-type glutamate receptors, we found low levels of BMAA to selectively damage vulnerable sub-populations of neurons, including motor neurons, via activation of AMPA/kainate receptors. Recent reports that BMAA is produced by cyanobacteria in diverse ecosystems and is present in brain and spinal cord tissues from sporadic ALS and Alzheimer's patients as well as brains of ALS/PDC patients provide strong motivation for further investigations of its toxic mechanisms and contributions to human disease.

The discovery of BMAA, and examples of biomagnification and protein incorporation involving other non-protein amino acids

Because of the similarity of ALS/PDC symptoms to those of the paralytic disease lathyrism, cycad seeds from Guam were analyzed for the presence of the non-protein amino acid b-ODAP, which is known to cause lathyrism. Although b-ODAP was not detected, a novel non-protein amino acid, now known as BMAA, was isolated. Primates are more sensitive to BMAA than rodents. It is possible that BMAA when ingested at a low concentration over a long period might be responsible for ALS/PDC. Some non-protein amino acids, including domoic acid and indospicine, are known to be biomagnified. Other non-protein amino acids including azetidine-2-carboxylic acid, canavanine, and selenium containing analogues of cystine and methionine have been shown to be misincorporated into proteins. Perhaps BMAA will not be the only non-protein amino acid that will be found in the brain tissues of those who died of a neurological disease.

Previous studies underestimate BMAA concentrations in cycad flour

The traditional diet of the Chamorro people of Guam has high concentrations of the neurotoxin BMAA, beta-methyl-amino-L-alanine, in cycad tortillas and from animals that feed on cycad seeds. We measured BMAA concentration in washed cycad flour and compared different extraction methods used by previous researchers in order to determine how much BMAA may have been unaccounted for in prior research. Samples were analyzed with AQC precolumn derivatization using HPLC-FD detection and verified with UPLC-UV, UPLC-MS, and triple quadrupole LC/MS/MS. Although previous workers had studied only the free amino acid component of BMAA in washed cycad flour, we detected significant levels of protein-associated BMAA in washed cycad flour. These data support a link between ALS/PDC and exposure to BMAA.

Purification, characterization, and sequencing of antimicrobial peptides, Cy-AMP1, Cy-AMP2, and Cy-AMP3, from the Cycad ( Cycas revoluta) seeds

Novel antimicrobial peptides (AMP), designated Cy-AMP1, Cy-AMP2, and Cy-AMP3, were purified from seeds of the cycad ( Cycas revoluta) by a CM cellulofine column, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They had molecular masses of 4583.2 Da, 4568.9 Da and 9275.8 Da, respectively, by MALDI–TOF MS analysis. Half of the amino acid residues of Cy-AMP1 and Cy-AMP2 were cysteine, glycine and proline, and their sequences were similar. The sequence of Cy-AMP3 showed high homology to various lipid transfer proteins. For Cy-AMP1 and Cy-AMP2, the concentrations of peptides required for 50% inhibition (IC 50) of the growth of plant pathogenic fungi, Gram-positive and Gram-negative bacteria were 7.0–8.9 μg/ml. The Cy-AMP3 had weak antimicrobial activity. The structural and antimicrobial characteristics of Cy-AMP1 and Cy-AMP2 indicated that they are a novel type of antimicrobial peptide belonging to a plant defensin family.

Determination of the neurotoxin BMAA (β-N-methylamino-l-alanine) in cycad seed and cyanobacteria by LC-MS/MS (liquid chromatography tandem mass spectrometry)

A highly specific method for the analysis of beta-N-methylamino-L-alanine (BMAA) by LC-MS/MS (liquid chromatography tandem mass spectrometry) has been developed and applied for cycad seeds and cyanobacteria. BMAA was analysed as a free fraction or as total BMAA after acidic hydrolysis to release any protein-bound BMAA. Deuterium labelled BMAA was synthesised and used as internal standard. The method comprises HILIC (hydrophilic interaction chromatography) and positive electrospray ionisation of the native compound, i.e. no derivatisation was used. For safe identification five specific product ions (m/z 102, 88, 76, 73 and 44), all derived from a precursor ion of m/z 119 and originating from different parts of the molecule, were detected (typical relative abundance 100%, 16%, 14%, 12% and 22% respectively). Cyanobacteria or muscle tissue was spiked with BMAA (10 to 1000 microg g(-1)) to validate the method (accuracy 95% to 109%, relative standard deviation 1% to 6%). The detection limit for free and total BMAA in tissue was <1 microg g(-1) and <4 microg g(-1) respectively. BMAA was successfully identified and quantified in cycad seeds, whereas previously reported findings of BMAA in samples of cyanobacteria could not be confirmed. Instead, the presence of alpha-,gamma-diamino butyric acid (DAB), an isomer of BMAA, was confirmed in one sample. The possible implications of this finding are discussed.

Analysis of dencichine in Panax notoginseng by gas chromatography–mass spectrometry with ethyl chloroformate derivatization

Dencichine (β- N-oxalyl- l-α,β-diaminopropionic acid) is a haemostatic agent present in well-known traditional Chinese medicinal herbs such as Panax notoginseng, as well as other Panax species. It is also a reported neurotoxic agent found in Lathyrus sativus (grass pea seed) and cycad seeds. A method was developed for quantitative determination of the non-protein amino acid, dencichine, in plant samples of P. notoginseng and the adventitious roots directly from the explants of P. notoginseng after derivatization with ethyl chloroformate (ECF) by gas chromatography–mass spectrometry (GC–MS). l-2-chlorophenylalanine was used as an internal standard. Calibration curves were linear ( r 2 = 0.9988, n = 6) in the range of 10–800 μg/ml for dencichine. Limit of detection and quantification for dencichine were 0.5 μg/ml and 2 μg/ml, respectively. This rapid and specific method may be applied to the quantification of dencichine in complex medicinal plants and their products.

From Cycad Flour, a New Suspect Emerges

NEURODEGENERATIVE DISEASEOne theory behind the strange neurological disorder on Guam holds that fruit bats concentrate a compound called BMAA in cycad seeds and deliver a whopping dose to anyone who eats the animals ([see main text][1]). Now, researchers have fingered a different suspect compound in cycad seeds, one that the native Chamorros of Guam ingest directly. ([Read more][2].) [1]: http://www.sciencemag.org/cgi/content/short/313/5786/428 [2]: http://www.sciencemag.org/cgi/content/full/313/5786/431

BMAA selectively injures motor neurons via AMPA/kainate receptor activation

The toxin beta-methylamino- l-alanine (BMAA) has been proposed to contribute to amyotrophic lateral sclerosis–Parkinsonism Dementia Complex of Guam (ALS/PDC) based on its ability to induce a similar disease phenotype in primates and its presence in cycad seeds, which constituted a dietary item in afflicted populations. Concerns about the apparent low potency of this toxin in relation to estimated levels of human ingestion led to a slowing of BMAA research. However, recent reports identifying potential new routes of exposure compel a re-examination of the BMAA/cycad hypothesis. BMAA was found to induce selective motor neuron (MN) loss in dissociated mixed spinal cord cultures at concentrations (∼ 30 μM) significantly lower than those previously found to induce widespread neuronal degeneration. The glutamate receptor antagonist NBQX prevented BMAA-induced death, implicating excitotoxic activation of AMPA/kainate receptors. Using microfluorimetric techniques, we further found that BMAA induced preferential [Ca 2+] i rises and selective reactive oxygen species (ROS) generation in MNs with minimal effect on other spinal neurons. Cycad seed extracts also triggered preferential AMPA/kainate-receptor-dependent MN injury, consistent with the idea that BMAA is a crucial toxic component in this plant. Present findings support the hypothesis that BMAA may contribute to the selective MN loss in ALS/PDC.

Neurotoxic flying foxes as dietary items for the Chamorro people, Marianas Islands

Fanihi – flying foxes ( Pteropus mariannus mariannus, Pteropodidae) – are a highly salient component of the traditional Chamorro diet. A neurotoxic, non-protein amino acid, β-methylamino- l-alanine (BMAA) accumulates in flying foxes, which forage on the seeds of Cycas micronesica (Cycadaceae) in Guam's forests. BMAA occurs throughout flying fox tissues both as a free amino acid and in a protein-bound form. It is not destroyed by cooking. Protein-bound BMAA also remains in cycad flour which has been washed and prepared by the Chamorro people as tortillas, dumplings, and thickened soups. Other animals that forage on cycad seeds may also provide BMAA inputs into the traditional Chamorro diet.

A link between ALS and short residence on Guam

Between 1940 and 1965, an unusually high incidence of ALS with co-occurrence in patients and families of a parkinsonism-dementia complex (PDC) was observed in the Chamorro population of Guam. So far, no genetic causes for ALS/PDC have been found. The increased risk of ALS in migrants to and from Guam suggests a combined effect of an environmental exposure and a genetic susceptibility.1,2 The declining incidence of ALS on Guam has been attributed to changes in the consuming patterns of food containing the seed of the locally growing cycad palms. These seeds were either processed traditionally in cycad flour or ingested indirectly by the consumption of indigenous flying foxes. These foxes were foraging on the cycad seeds and are supposed to contain high concentrations of the neurotoxic components of the seeds.3,4 We reviewed information on residence on Guam in a previously conducted case-control study of ALS and investigated the possibility that residence in Guam contributed to the risk for ALS in citizens of the United States. The study population …

A mechanism for slow release of biomagnified cyanobacterial neurotoxins and neurodegenerative disease in Guam.

As root symbionts of cycad trees, cyanobacteria of the genus Nostoc produce beta-methylamino-l-alanine (BMAA), a neurotoxic nonprotein amino acid. The biomagnification of BMAA through the Guam ecosystem fits a classic triangle of increasing concentrations of toxic compounds up the food chain. However, because BMAA is polar and nonlipophilic, a mechanism for its biomagnification through increasing trophic levels has been unclear. We report that BMAA occurs not only as a free amino acid in the Guam ecosystem but also can be released from a bound form by acid hydrolysis. After first removing free amino acids from tissue samples of various trophic levels (cyanobacteria, root symbioses, cycad seeds, cycad flour, flying foxes eaten by the Chamorro people, and brain tissues of Chamorros who died from amyotrophic lateral sclerosis/Parkinsonism dementia complex), we then hydrolyzed the remaining fraction and found BMAA concentrations increased 10- to 240-fold. This bound form of BMAA may function as an endogenous neurotoxic reservoir, accumulating and being transported between trophic levels and subsequently being released during digestion and protein metabolism. Within brain tissues, the endogenous neurotoxic reservoir can slowly release free BMAA, thereby causing incipient and recurrent neurological damage over years or even decades, which may explain the observed long latency period for neurological disease onset among the Chamorro people. The presence of BMAA in brain tissues from Canadian patients who died of Alzheimer's disease suggests that exposure to cyanobacterial neurotoxins occurs outside of Guam.

Conservation Implications of Chamorro Consumption of Flying Foxes as a Possible Cause of Amyotrophic Lateral Sclerosis–Parkinsonism Dementia Complex in Guam

Abstract: In the Pacific island of Guam, flying fox ( Pteropus mariannus and P. tokudae ) populations historically persisted for centuries despite subsistence foraging by the indigenous Chamorro people. However, the militarization of Guam in the twentieth century, with an increased emphasis on a cash rather than a subsistence economy, led to two features that had deleterious effects on the flying fox populations: easy access to firearms greatly increased hunter yields and disposable income facilitated commercial traffic in flying foxes. As a result, consumption of flying foxes dramatically increased, leading to the extinction of one indigenous species and near extinction of another. The demise of the flying fox populations was mirrored by a subsequent decrease in the health of the Chamorro people. Amyotrophic lateral sclerosis–Parkinsonism dementia complex ( ALS‐PDC ), a neurological disease with aspects of ALS, Alzheimer's, and Parkinson's disease, became a major cause of adult death in some villages. It has been recently hypothesized that consumption of flying foxes by the Chamorro may have generated sufficiently high cumulative doses of neurotoxins to result in the observed high levels of ALS‐PDC among the Chamorros in Guam because flying foxes forage on cycad seeds that are rich in neurotoxins. Commercialization of traffic in wild flying fox populations may have therefore produced grave health consequences for the Chamorro people.