Chemometric Analysis of Cannabinoids: Chemotaxonomy and Domestication Syndrome.

Cannabis is an interesting domesticated crop with a long history of cultivation and use. Strains have been selected through informal breeding programs with undisclosed parentage and criteria. The term "strain" refers to minor morphological differences and grower branding rather than distinct cultivated varieties. We hypothesized that strains sold by different licensed producers are chemotaxonomically indistinguishable and that the commercial practice of identifying strains by the ratio of total THC and CBD is insufficient to account for the reported human health outcomes. We used targeted metabolomics to analyze 11 known cannabinoids and an untargeted metabolomics approach to identify 21 unknown cannabinoids. Five clusters of chemotaxonomically indistinguishable strains were identified from the 33 commercial products. Only 3 of the clusters produce CBDA in significant quantities while the other 2 clusters redirect metabolic resources toward the THCA production pathways. Six unknown metabolites were unique to CBD-rich strains and/or correlated to CBDA and 3 unknowns were found only in THC-rich strains. Together, these data indicate the domestication of the cannabis germplasm has resulted in a loss of the CBDA pathway in some strains and reallocation of resources between CBDA and THCA pathways in others. The impact of domestication is a lack of chemical diversity and loss of biodiversity in modern cannabis strains.

Simultaneous induction of jasmonic acid and disease-responsive genes signifies tolerance of American elm to Dutch elm disease.

Dutch elm disease (DED), caused by three fungal species in the genus Ophiostoma, is the most devastating disease of both native European and North American elm trees. Although many tolerant cultivars have been identified and released, the tolerance mechanisms are not well understood and true resistance has not yet been achieved. Here we show that the expression of disease-responsive genes in reactions leading to tolerance or susceptibility is significantly differentiated within the first 144 hours post-inoculation (hpi). Analysis of the levels of endogenous plant defense molecules such as jasmonic acid (JA) and salicylic acid (SA) in tolerant and susceptible American elm saplings suggested SA and methyl-jasmonate as potential defense response elicitors, which was further confirmed by field observations. However, the tolerant phenotype can be best characterized by a concurrent induction of JA and disease-responsive genes at 96 hpi. Molecular investigations indicated that the expression of fungal genes (i.e. cerato ulmin) was also modulated by endogenous SA and JA and this response was unique among aggressive and non-aggressive fungal strains. The present study not only provides better understanding of tolerance mechanisms to DED, but also represents a first, verified template for examining simultaneous transcriptomic changes during American elm-fungus interactions.

Artemisia judaica L.: micropropagation and antioxidant activity.

Artemisia judaica L., an Egyptian medicinal plant used in the treatment of gastrointestinal disorders, was mass-propagated and grown using solid, paper-bridge-support liquid, liquid-flask and bioreactor cultures. The liquid-flask culture using 50 ml MS liquid medium in 250 ml flask yielded significantly greater shoot proliferation than either solid cultures or paper-bridge-support liquid cultures. Increasing flask capacity from 100 to 500 ml improved shoot proliferation and growth. Mass-propagation efficiencies of various bioreactor systems, viz. temporary immersion reactors and bubble column reactors, were also compared. The temporary immersion bioreactor was found to have significant advantages for A. judaica shoot proliferation. The shoot cultures from the temporary immersion reactor formed complete plantlets when subcultured onto a medium containing 1 micromoll(-1) indole-3-butyric acid (IBA), and mature plants were established, acclimatized and thrived in standard greenhouse conditions. Assays of antioxidant activity and total flavonoid content of in vitro and in vivo grown tissues were evaluated as gross parameters of medicinal efficacy. Significantly higher antioxidant activity and flavonoid contents were observed in the tissues of mature greenhouse-grown plants. The efficient in vitro production systems developed in this study provided sterile, consistent tissues for investigation of bioactivity and germplasm conservation of A. judaica.

Tryptophan is a precursor for melatonin and serotonin biosynthesis in in vitro regenerated St. John's wort (Hypericum perforatum L. cv. Anthos) plants.

HYPERICUM PERFORATUM: cv. Anthos) is presented. Isotope tracer experiments were performed on plantlets regenerated from thidiazuron-induced stem explants and grown on MS basal medium for 2 months. Radiolabel from 14C-tryptophan was recovered as 14C-indoleacetic acid, 14C-tryptamine, 14C-5-hydroxytryptophan, 14C-serotonin and 14C-melatonin in the treated St. John's wort plantlets. Chromatographic peak identity was confirmed by high performance liquid chromatography-mass spectrometry-mass spectrometry and quantification of melatonin by radioimmunoassay. Significantly more radiolabel was recovered in serotonin relative to melatonin under low light conditions with this ratio being reversed under increased lighting, indicating that the rate of flow through this biosynthetic pathway is regulated, at least in part, by light.

Thidiazuron-induced plant regeneration from hypocotyl cultures of St. John's wort (Hypericum perforatum. cv 'Anthos').

St. John's wort (Hypericum perforatum. cv 'Anthos') is a medicinal plant with evidence of efficacy as an anti-depressant. The present report describes the development of an in vitro regeneration system that utilizes thidiazuron [N-phenyl-N'-(1,2,3-thidiazol-yl)urea] for the induction of de novo shoots on etiolated hypocotyl segments of St. John's wort seedlings. The optimum level of thidiazuron supplementation to the culture medium was 5 µmol·l-1 for a 9-day induction period followed by subculture of induced hypocotyl explants on basal medium. Other plant growth regulators including benzyladenine and indoleacetic acid were not effective in inducing regeneration on St. John's wort hypocotyls. Histological examination of the cultures revealed that the regenerated plants were derived from de novo developed shoots. Transfer of the regenerated shoots into a liquid medium with no plant growth regulators resulted in the rapid and prolific growth of viable plantlets. The rapid and efficient micropropagation system for St. John's wort may be useful for both the genetic improvement of this crop and the production of high-quality phytopharmaceutical preparations for the treatment of neurological disorders.

Proline is synthesized from glutamate during intragastric infusion but not during intravenous infusion in neonatal piglets.

Glutamate is considered the primary precursor amino acid for proline synthesis in mammals. Evidence exists, however, suggesting that proline may be a dietary indispensable amino acid for 2.5-kg piglets due to inadequate synthesis. This hypothesis was tested by intravenous and intragastric infusion of radiolabeled amino acids in vivo. Piglets (3 to 4 d old) were surgically implanted with catheters in the femoral (infusion) and jugular (sampling) veins and in the stomach (feeding and infusion). Piglets were fed hourly, via the stomach catheter, a semi-purified diet containing 10% dried skim milk, 15% corn oil, amino acids, vitamins and minerals. Experiment 1 was a 2 X 2 factorial design, with 24 piglets adapted to either low or supplemental proline diets (1.3 and 16.4 g proline x kg(-1) respectively) for 7 d, then intravenously infused with either [U-14C]glutamate or [U-14C]proline (185 k Bq x kg(-1) prime; 370 kBq x kg(-1) x h(-1) constant) for 4 h. Experiment 2 followed similar protocols, with eight piglets adapted to the low proline diet for 7 d and [U-14C]glutamate or [U-14C]proline infused into the stomach catheter. Piglets infused intravenously with [U-14C]glutamate did not convert glutamate to proline. Radioactive label was recovered in proline in all of the piglets receiving intragastric infusion of [U-14C]-glutamate. The fractional synthesis rate of proline from intragastric glutamate was 125 microgram ol kg(-1) x h(-1), accounting for approximately 40% of the proline accumulated. These data provide conclusive evidence that intravenously infused glutamate is not used as a precursor for proline synthesis and that, although conversion of glutamate to proline occurs in the gastrointestinal tract, the rate is not sufficient to provide the proline accumulated.

Regeneration of the Egyptian medicinal plant Artemisia judaica L.

An in vitro propagation system for Artemisia judaica L., a traditional Egyptian medicinal plant, has been developed. De novo shoot organogenesis was induced by culturing etiolated hypocotyls and intact seedlings on medium supplemented with thidiazuron [N-phenyl-N'-(1,2,3-thidiazol-yl) urea] via callusing at the cotyledonary notch region. Up to 16 shoots formed per seedling cultured on a medium containing 1 micro mol l(-1) thidiazuron for an optimal duration of exposure of 20 days. Regenerated shoots formed roots when subcultured onto a medium containing 1 micromol l(-1) indole-3-butyric acid. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of A. judaica.

Morphoregulatory role of thidiazuron: morphogenesis of root outgrowths in thidiazuron-treated geranium (Pelargonium x hortorum Bailey).

Root outgrowths formed on the root tissue of geranium (Pelargonium x hortorum Bailey cv. Kim and cv. Shone Helena) plants in response to treatment with the phenylurea derivative, thidiazuron (N-phenyl-N'-1,2,3-thiadiazol-5'-ylurea; TDZ). Treatment with the cytokinin N(6)-benzylaminopurine (BAP) or the auxin α-naphthaleneacetic acid (NAA) did not result in stimulation of similar abnormal structures on the root tissue. Significantly more outgrowths developed on roots of plants treated with 10 µM and 20 µM TDZ than on control plants or those treated with 1 µM TDZ for the eight-week treatment period. Some outgrowths produced shoots and plantlets while still attached to roots, and regenerants were easily separated from the root tissue and transferred to soil in the greenhouse where they grew to maturity. Histological observations suggested these outgrowths originated from the vascular cambium region of the root.

Occurrence of beta-methylamino-l-alanine (BMAA) in ALS/PDC patients from Guam.

We tested the brain tissues of the Chamorro people of Guam who died of amyotrophic lateral sclerosis/Parkinsonism dimentia complex (ALS/PDC) for the neurotoxin beta-methylamino-l-alanine (BMAA). We used validated high-pressure liquid chromatography and liquid chromatography-mass spectrometry analyses to test well-characterized archival tissues of the superior frontal gyrus from eight Chamorros from Guam and a comparison group of 15 Canadians. BMAA was found as a free amino acid in 83% of Chamorro ALS/PDC patients (3-10 microg/g) as a protein-associated amino acid in 100% of the Chamorro individuals (149-1190 microg/g). Both forms of BMAA were also found at comparable levels in two Canadians who died of progressive neurodegenerative disease. BMAA, which is produced by cyanobacteria, may be associated with some cases of neurodegenerative disease.