Tag: DNA

Cannabinoid Receptor 2 Functional Variant Contributes to the Risk for Pediatric Inflammatory Bowel Disease.

Strisciuglio C1, Bellini G, Miele E, Martinelli M, Cenni S, Tortora C, Tolone C, Miraglia Del Giudice E, Rossi F.

Journal Of Clinical Gastroenterology, 2018

DOI: 10.1097/MCG.0000000000000755

We conducted a case-control association analysis to establish the role of a common CB2 functional variant, Q63R, in the susceptibility to inflammatory bowel disease (IBD). We genotyped 217 pediatric IBD patients [112 Crohn’s disease (CD), 105 ulcerative colitis (UC)] and 600 controls for the CB2-Q63R variant by Taqman assay. Data were collected from clinical records on age at diagnosis, disease activity, duration and location, extraintestinal manifestations, therapy, clinical relapses, and need for surgery.
Goals & Study

Cannabis plants having modified expression of thca synthase

Patent: WO2016189384A1 
Inventor Ekaterina Alexandra Boudko, Thomas Shipley IV, Douglas Johnson
 https://patents.google.com/patent/WO2016189384A1/en

The invention relates to novel genetically modified plants and methods or materials, such as polynucleotides, expression cassettes, or vectors for producing the same. Moreover, the invention relates to altering the content of cannabinoids in plants and to medical compositions derived from such plants. In particular embodiments, the present invention relates to cannabis plants having modified expression of tetrahydrocannabinolic acid (THCA) synthase and methods of modifying the amount of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in cannabis by modifying expression of THCA synthase.
Abstract

The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acylactivating enzyme in Cannabis sativa trichomes

Jake M Stout, Zakia Boubakir, Stephen J Ambrose, Jonathan E Page

February 2012 The Plant Journal 71(3):353-65 

DOI: 10.1111/j.1365-313X.2012.04949.x

The psychoactive and analgesic cannabinoids (e.g. Δ(9) -tetrahydrocannabinol (THC)) in Cannabis sativa are formed from the short-chain fatty acyl-coenzyme A (CoA) precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g(-1) fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes that encoded putative AAEs including CsAAE1, which shows high transcript abundance in glandular trichomes. In vitro assays showed that recombinant CsAAE1 activates hexanoate and other short- and medium-chained fatty acids. This activity and the trichome-specific expression of CsAAE1 suggest that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis showed that CsAAE1 groups with peroxisomal AAEs, it lacked a peroxisome targeting sequence 1 (PTS1) and localized to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome expressed sequence tag (EST) dataset for enzymes of fatty acid metabolism. The high abundance of transcripts that encode desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway that involves the oxygenation and breakdown of unsaturated fatty acids.
Abstract

*Qualitative and Quantitative Aspects of the Inheritance of Chemical Phenotype in Cannabis

Giuseppe Mandolino, Manuela Bagatta, Andrea Carboni, Paolo Ranalli, Etienne de Meijer

March 2003 Journal of Industrial Hemp 8(2):51-72

DOI: 10.1300/J237v08n02_04

Four crosses were made between Cannabis plants with “pure” CBD and THC chemotypes. The F1 plants obtained were self-fertilised to produce F2s. Chemotypical distributions were analysed by gas-chromatography. A segregation analysis of the different F2 progenies obtained showed that chemotype, estimated as CBD/THC ratio, behaves as a qualitative character, and a model for a single locus B, with two co-dominant alleles, BD and BT is described. The CBD/THC ratios in the F1 offsprings were found to be significantly different in the heterozygous plants from the different pedigrees. The amount of CBD plus THC in the same pedigrees was also described. Heterosis was found to be a common feature, but not a general one, of cannabinoid accumulation in the F1s. Distribution of the values of cannabinoid content in classes was found to be normal. RAPD markers linked to the segregating chemotypes (“pure” CBD and “pure” THC) were identified by bulk segregant analysis, and the degree of linkage of these markers with the chemotype was described.
Abstract

Pharmacogenetics of Cannabinoids

Szymon Hryhorowicz, Michal Walczak, Oliwia Zakerska-Banaszak, Ryszard Słomski, and Marzena Skrzypczak-Zielińska

Eur J Drug Metab Pharmacokinet. 2018; 43(1): 1–12.

doi: 10.1007/s13318-017-0416-z

Although the application of medical marijuana and cannabinoid drugs is controversial, it is a part of modern-day medicine. The list of diseases in which cannabinoids are promoted as a treatment is constantly expanding. Cases of significant improvement in patients with a very poor prognosis of glioma or epilepsy have already been described. However, the occurrence of side effects is still difficult to estimate, and the current knowledge of the therapeutic effects of cannabinoids is still insufficient. In our opinion, the answers to many questions and concerns regarding the medical use of cannabis can be provided by pharmacogenetics. Knowledge based on proteins and molecules involved in the transport, action, and metabolism of cannabinoids in the human organism leads us to predict candidate genes which variations are responsible for the presence of the therapeutic and side effects of medical marijuana and cannabinoid-based drugs. We can divide them into: receptor genes—CNR1, CNR2, TRPV1, and GPR55, transporters—ABCB1, ABCG2, SLC6A, biotransformation, biosynthesis, and bioactivation proteins encoded by CYP3A4, CYP2C19, CYP2C9, CYP2A6, CYP1A1, COMT, FAAH, COX2, ABHD6, ABHD12 genes, and also MAPK14. This review organizes the current knowledge in the context of cannabinoids pharmacogenetics according to individualized medicine and cannabinoid drugs therapy
Abstract

Identification of candidate genes affecting Δ9-tetrahydrocannabinol biosynthesis in Cannabis sativa

Marks MD, Tian L, Wenger JP, Omburo SN, Soto-Fuentes W, He J, Gang DR, Weiblen GD, Dixon RA (2009)

J Exp Bot 60: 3715–3726

doi: 10.1093/jxb/erp210

RNA isolated from the glands of a D9-tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands.
Abstract

Identification and Characterization of Cannabinoids That Induce Cell Death through Mitochondrial Permeability Transition in Cannabis Leaf Cells

Satoshi Morimoto 1 , Yumi Tanaka, Kaori Sasaki, Hiroyuki Tanaka, Tomohide Fukamizu, Yoshinari Shoyama, Yukihiro Shoyama and Futoshi Taura 

2007 The Journal of Biological Chemistry, 282, 20739-20751.

DOI: 10.1074/jbc.M700133200

Cannabinoids are secondary metabolites stored in capitate-sessile glands on leaves of Cannabis sativa. We discovered that cell death is induced in the leaf tissues exposed to cannabinoid resin secreted from the glands, and identified cannabichromenic acid (CBCA) and Δ1-tetrahydrocannabinolic acid (THCA) as unique cell death mediators from the resin. These cannabinoids effectively induced cell death in the leaf cells or suspension-cultured cells of C. sativa, whereas pretreatment with the mitochondrial permeability transition (MPT) inhibitor cyclosporin A suppressed this cell death response. Examinations using isolated mitochondria demonstrated that CBCA and THCA mediate opening of MPT pores without requiring Ca2+ and other cytosolic factors, resulting in high amplitude mitochondrial swelling, release of mitochondrial proteins (cytochrome c and nuclease), and irreversible loss of mitochondrial membrane potential. Therefore, CBCA and THCA are considered to cause serious damage to mitochondria through MPT. The mitochondrial damage was also confirmed by a marked decrease of ATP level in cannabinoid-treated suspension cells. These features are in good accord with those of necrotic cell death, whereas DNA degradation was also observed in cannabinoid-mediated cell death. However, the DNA degradation was catalyzed by nucleases released from mitochondria during MPT, indicating that this reaction was not induced via a caspase-dependent apoptotic pathway. Furthermore, the inhibition of the DNA degradation only slightly blocked the cell death induced by cannabinoids. Based on these results, we conclude that CBCA and THCA have the ability to induce necrotic cell death via mitochondrial dysfunction in the leaf cells of C. sativa.
Abstract

Gene duplication and divergence affecting drug content in Cannabis sativa

George D. Weiblen, Jonathan P. Wenger, Kathleen J. Craft, Mahmoud A. ElSohly, Zlatko Mehmedic, Erin L. Treiber, M. David Marks

New Phytologist 17 July 2015

DOI: 10.1111/nph.13562

Cannabis sativa is an economically important source of durable fibers, nutritious seeds, and psychoactive drugs but few economic plants are so poorly understood genetically. Marijuana and hemp were crossed to evaluate competing models of cannabinoid inheritance and to explain the predominance of tetrahydrocannabinolic acid (THCA) in marijuana compared with cannabidiolic acid (CBDA) in hemp. Individuals in the resulting F2 population were assessed for differential expression of cannabinoid synthase genes and were used in linkage mapping. Genetic markers associated with divergent cannabinoid phenotypes were identified. Although phenotypic segregation and a major quantitative trait locus (QTL) for the THCA/CBDA ratio were consistent with a simple model of codominant alleles at a single locus, the diversity of THCA and CBDA synthase sequences observed in the mapping population, the position of enzyme coding loci on the map, and patterns of expression suggest multiple linked loci. Phylogenetic analysis further suggests a history of duplication and divergence affecting drug content. Marijuana is distinguished from hemp by a nonfunctional CBDA synthase that appears to have been positively selected to enhance psychoactivity. An unlinked QTL for cannabinoid quantity may also have played a role in the recent escalation of drug potency.
Abstract

Differential transcriptional profiles mediated by exposure to the cannabinoids cannabidiol and D9 -tetrahydrocannabinol in BV-2 microglial cells

Ana Juknat, Maciej Pietr, Ewa Kozela1, Neta Rimmerman, Rivka Levy, Giovanni Coppola, Daniel Geschwind and Zvi Vogel

British Journal of Pharmacology (2012) 165 2512–2528

DOI: 10.1111/j.1476-5381.2011.01461.x

Apart from their effects on mood and reward, cannabinoids exert beneficial actions such as neuroprotection and attenuation of inflammation. The immunosuppressive activity of cannabinoids has been well established. However, the underlying mechanisms are largely unknown. We previously showed that the psychoactive cannabinoidD9 -tetrahydrocannabinol (THC) and the non-psychoactive cannabidiol (CBD) differ in their anti-inflammatory signalling pathways.
Background and Purpose

Characterisation of cannabinoid composition in a diverse Cannabis sativa L. germplasm collection

Matthew T. Welling, Lei Liu, Tim Shapter, Carolyn A. Raymond, Graham J. King
Euphytica (2016) 208:463–475
DOI: 10.1007/s10681-015-1585-y

The ability to characterise cannabinoid chemical phenotype (chemotype) accurately is important for the development of Cannabis sativa L. cultivars specific for pharmacological, hemp fibre, or seed end use. Although a number of chemotyping and genotyping methods have previously been developed to predict and characterise cannabinoid composition, only a subset of the gene pool has been examined. A representative survey from a wide range of geographically and genetically diverse C. sativa accessions using liquid chromatography–mass spectrometry (LC–MS) cannabinoid profiling together with dominant and co-dominant DNA marker assays was performed. Overall variability of chemotype across the gene pool was found to be three-fold greater within heterozygote genotypes than previously reported. Interestingly, an individual plant of East Asian origin was found to exhibit a rare propyl alkyl cannabinoid homologue and a chemotype inconsistent with the predicted genotype. We propose that in order to carry out comprehensive screening of genetic resource collections and to identify chemotypic variants specific for end-use pharmacological applications, a strategy which adopts both cannabinoid profiling and the co-dominant DNA marker assay is required. Further research with consideration of propyl-alkyl-cannabinoid homologues should explore the relationship between chemotype and genotype in greater detail.
Abstract